JPH08122648A - Differential interference microscope - Google Patents

Abstract

PURPOSE: To provide a differential interference microscope which can obtain optimum resolving power in combination with a contrast increasing method. CONSTITUTION: The differential interference microscope which separates the light from a light source 7 into ordinary light and extraordinary light to irradiate a body 1 to be observed and then forms an image of the body 1 to be observed by putting the ordinary light and extraordinary light one over the other through an image formation optical system 3 has an electron image pickup element 4 arranged on the image formation surface of the image formation optical system 3, and Δ<0.25.λ/NA is satisfied, where Δ is the separation width of the ordinary light and extraordinary light, irradiating the body 1 to be observed, on the body surface, λ is the wavelength of the observed light, and the NA is the numerical aperture of the image formation optical system 3 on the side of the body to be observed, and the image signal from the electron image pickup element 4 is processed to increase the contrast.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a differential interference microscope used for observing fine objects such as cells and bacteria with high resolution.

[0002]

2. Description of the Related Art In recent years, an optical microscope has been often used to observe the fine structure of living cells and the response to external stimuli such as bacteria. Therefore, an optical microscope having a higher resolution has been demanded. There is.

Under these circumstances, various methods for improving the resolving power of a microscope have been proposed, and one method is, for example, "Video-Enhanced Contrast, Differe.
ntial Interference Contrast (AVEC-DIC) Microscopy:
A New Method Capable of Analyzing Microtubule-Rela
ted Motility in the Reticulopodial Network of Allo
gromia Iaticollaris '' RDAllen, NSAllen, JLTr
avis, Cell Motility1: 291-302 (1981), "Recent Advances in Optical Microscopy Systems" Takeshi Hayakawa, Shuji Fujibun, Journal of the Physical Society of Japan, Volume 40, No. 1, P.47-55 (1985), etc. Contrast enhancement method is disclosed. By using this contrast enhancement method, even an image that is difficult to identify due to low contrast by conventional visual observation can be identified by enhancing the cost last.

The contrast enhancement method is often combined with a differential interference microscope, and is effective in increasing the resolution of the differential interference microscope.

[0005]

In the differential interference microscope, the width of separation between the ordinary light and the extraordinary light on the observation object surface is called the shear amount, and this shear amount is the resolution or contrast of the differential interference microscope. It is known to be an important parameter that determines

However, the determination of the amount of shear in the conventional differential interference microscope is not based on theoretical analysis,
It is determined by empirical process so that it looks best by visual observation. In the above two documents, no theoretical consideration has been given to the share amount.

Also, "Limits of Resolution of Optical Microscopes" Hiroshi Oki, Proc. Of Super Resolution Seminar, Japan Spectroscopy Society, P.
1-15 (1992) and "Modulation Transfar Function" S. I
noue, VIDEO MICROSCOPY, Plenum publising CO, 5.7,
In p122-125, the imaging characteristics of a differential interference microscope are analyzed, but in these documents, there is no mention of the influence of the shear amount on the imaging characteristics.

As described above, in the conventional differential interference microscope, it has been attempted to realize a high resolution by combining the contrast enhancement method, but since the share amount is not taken into consideration, There is a problem that the optimum resolution cannot always be obtained.

The present invention has been made in view of the above points, and an object of the present invention is to provide a differential interference microscope which is appropriately configured so as to obtain an optimum resolving power in combination with a contrast enhancement method.

[0010]

To achieve the above object, the present invention separates light from a light source into ordinary light and extraordinary light, irradiates the observed object, and forms an image of the observed object. In a differential interference microscope in which ordinary light and extraordinary light are superposed by an optical system to form an image on an image forming surface, an electronic image pickup element is arranged on the image forming surface of the image forming optical system, and the object to be observed is irradiated. Assuming that the separation width Δ of the ordinary light and the extraordinary light in the object plane is λ, where the wavelength of the light to be observed is NA, and the numerical aperture on the observation object side of the imaging optical system is NA, Δ <0.25.
The image signal from the electronic image pickup device is processed so as to satisfy λ / NA, and the contrast is enhanced.

The separation width Δ is 0.08 · λ / NA ≦ Δ
It is preferable to satisfy the above condition from the viewpoint of obtaining optimum resolution.

[0012]

FIG. 1 shows the construction of an example of a differential interference microscope for explaining the principle of the present invention. This differential interference microscope includes an illumination optical system 2 for illuminating a sample 1, and a sample 1
Image forming optical system 3 for enlarging and forming an image of, the electronic image pickup device 4 arranged on the image forming plane of the image forming optical system 3, and the image signal from the electronic image pickup device 4 is processed to increase the contrast. It has an image processing device 5 for enhancing and an output device 6 for displaying the image signal processed by the image processing device 5.

The illumination optical system 2 has a light source 7, a polarizer 8, a Nomarski prism 9 and a condenser lens 10. The light from the light source 7 is linearly polarized by the polarizer 8 and is incident on the Nomarski prism 9. The normal light and the extraordinary light are separated and the sample 1 is illuminated through the condenser lens 10. Further, the imaging optical system 3 includes the objective lens 11,
Having the Nomarski prism 12 and the analyzer 13, the ordinary and extraordinary rays that have passed through the sample 1 are combined by the Nomarski prism 12 via the objective lens 11, and the combined ordinary and extraordinary rays are interfered by the analyzer 13. Then, an interference image is formed on the image plane.

Imaging of the differential interference microscope shown in FIG. 1 will be described below using a one-dimensional model. Now, let the pupil function of the illumination optical system 2 be Q (ξ) and the pupil function of the imaging optical system 3 be P (ξ).
(ξ), the phase distribution of sample 1 is φ (x), and the Born approximation, exp i φ (x) = 1 + iφ (x), is performed, the image intensity distribution I (x) of sample 1 becomes From the coherent imaging theory,

[Equation 1] Given in. However, Φ (f) represents the Fourier transform of φ (x), and f represents the spatial frequency.

R (f, f ') in the above equation (1) represents a transfer function in partially coherent imaging, and the second and third terms are calculated when either f or f'is zero. Yes, the fourth term represents the calculation when f ≠ 0 and f ′ ≠ 0.

Here, since the differential interference microscope uses polarization interference, consider polarization coordinates in which the directions of vibration of ordinary and extraordinary rays in the Nomarski prism 12 are coordinate axis directions.

The light emitted from the light source 7 is linearly polarized by the polarizer 8 and enters the Nomarski prism 9.
Here, the ordinary light and the extraordinary light are separated into polarization components orthogonal to each other. Therefore, the pupil function P (ξ) of the imaging optical system 3 is represented by a vector, and P (ξ) = (a Px (ξ), b Py (ξ)). In addition, a and b represent the polarization components of the linearly polarized light by the polarizer 8.

The separated ordinary and extraordinary rays pass through two points which are separated from the sample 1 by the share amount Δ, pass through the objective lens 11 of the imaging optical system 3 and are combined by the Nomarski prism 12 and then detected. When they pass through the photons 13, they interfere with each other to form an interference image on the image plane. Therefore,
If the polarization components of the analyzer 13 are α and β,

[Equation 2] It is represented by.

In the above Px (ξ) and Py (ξ), the phase difference (retardation) between the ordinary ray and the extraordinary ray is θ, and the bright-field pupil function of the imaging optical system 3 is p (ξ). Then,

(Equation 3) Assuming that the polarizer 8 and the analyzer 13 are orthogonal to each other (crossed nicols) and the imaging optical system 3 is an ideal optical system, the above equation (1) is expressed by the above equations (2) and (3). )
Using the formula,

[Equation 4] Can be written as

If the sample 1 is not thick and the fourth term of the equation (4) can be ignored, the image intensity distribution Is (x) in the shear direction of the differential interference microscope is approximately

(Equation 5) And can be expressed as a combination of the bright field image and the differential interference image.

On the other hand, in the direction perpendicular to the shear direction,
Since the rays are not separated, if the image intensity distribution is Ib (x), then we can set Δ = 0 in equation (5),

(Equation 6) And only the brightfield image.

In the image observed by the differential interference microscope, the sample 1
The retardation amount is adjusted so that the direction of the phase gradient (upward gradient, downward gradient) is changed to a contrast of light and dark so that it looks three-dimensional. Therefore, if the retardation is adjusted so that θ = 0, the equations (5) and (6) become

(Equation 7) Therefore, only the differential interference image is obtained, and either the ascending slope or the descending slope in the shear direction becomes a bright image.

On the other hand, the retardation amount is θ ≠
When taking a relatively small value of 0, (5) and (6)
The formula is approximately

(Equation 8) It is represented by. From these equations (8) and (9), it is understood that the contrast in the differential interference microscope depends on the contrast in the shear direction.

From the above equation (8), the MTF representing the image forming characteristic in the differential interference microscope is MTF (f) = sin (fΔ / 2) ∫Q (ξ) p (ξ + f) p ^* (ξ) It is given by d ξ (10). From this equation (10), it can be seen that the imaging characteristic of the differential interference microscope depends on the shear amount.

Here, the pupil of the illumination optical system 2 and the imaging optical system 3
And the pupil are made circular with the same size, and the share amount is Δ = 0.61λ using a coordinate system in which the radius of the pupil is standardized to 1.
FIG. 2 shows the results of calculating the MTFs for / NA, 0.3λ / NA, and 0.15λ / NA. Figure 2
From this, it is understood that as the share amount is decreased, the resolution is increased, but the contrast is lowered. When the share amount is decreased by visual observation, the same result as that the contrast is deteriorated is obtained.

On the other hand, instead of the visual observation, the electronic image pickup device 4 is used.
By using a contrast enhancement method in which the image signal is processed by the image processing device 5 to enhance the contrast, the background brightness (DC component) of the image can be removed and only the contrast component can be enhanced. Therefore, it is possible to enhance an image even if the image has poor contrast. For example, an optical system or electrical noise obtained by previously defocusing the sample 1 is subtracted from the image signal of the electronic image pickup device 4, and an appropriate preset offset level (threshold value) is set from the subtracted signal. Extract the signal that exceeds and enhance it.

By using such a contrast enhancement method, the background of the observed object in the sample 1 of interest, the surrounding components having a relatively low spatial frequency, and the transmitted zero-order component can be reduced, so that the contrast is low. It is possible to enhance the contrast of the details that were not visible, and it is possible to clearly observe the details. For example, the intensity distribution of the image of the observed object when the contrast enhancement method is not applied is shown in FIG.
It is assumed that it was shown in. In FIG. 3, when the intensity change of the part of interest is x and the maximum value of the intensity distribution is X, the contrast is x / X. This contrast x
It is known that / X cannot be identified when it is about 20% or less.

Therefore, by using the contrast enhancement method, as shown in FIG.
Is converted into an image signal by the electronic image pickup device 4, and a noise component such as illumination unevenness is removed from the image signal by the image processing device 5, and then a signal X'exceeding an appropriately set threshold Y is taken out. Amplify. By doing so, as shown in FIG. 4, the maximum value of the signal is amplified to X ″ and the intensity of the portion of interest is amplified to x ′, and the contrast x ′ / X ″ exceeds 20%, It is possible to enhance the contrast of the part that could not be identified by normal visual observation,
It is possible to identify details.

Using the contrast enhancement method described above,
Equation (8) is

[Equation 9] It is represented by.

Therefore, since the standardization of MTF can be changed and the maximum value of MTF can be set to 1,
2 can be rewritten as shown in FIG. As is clear from FIG. 5, when the contrast enhancement method is used,
It can be seen that the resolution of the differential interference microscope becomes advantageous when the shear amount is reduced.

However, even if the share amount is reduced as much as possible, the resolution does not increase below a certain limit value. In view of this point, the present invention provides a shear amount that optimizes resolution when a contrast enhancement method is applied to a differential interference microscope. Hereinafter, this point will be described.

First, in order for the differential interference microscope to have the same limiting resolution as in normal bright field observation, the cutoff frequencies of both should be the same. This is (7) above
From the equation, it can be seen that when the shear amount Δ is Δ = 0.5λ / NA, the cutoff frequencies of the differential interference microscope and the bright field observation match. However, in this case, as shown in FIG. 6, the frequency characteristic in the vicinity of the cutoff becomes low, which makes it unsuitable for high resolution. Therefore, in order to increase the resolution of the differential interference microscope, it is necessary to improve the frequency characteristics near the cutoff.

Therefore, in the present invention, the shear amount Δ of the differential interference microscope is set to satisfy Δ <0.25 · λ / NA. By doing this, as shown in FIG.
MT in a differential interference microscope near the cutoff
The frequency characteristics of F can be improved to be the same as in bright field observation.

Next, the lower limit of the share amount Δ will be described. In FIG. 8, the share amount Δ is 0.08λ / NA, 0.0.
FIG. 9 shows normal MTF characteristics in the case of 7λ / NA and 0.06λ / NA in comparison with MTF characteristics in bright field observation. FIG. 9 shows contrast enhancement at each share amount Δ in FIG. MT when
The results of calculating the F characteristics are shown in comparison with the MTF characteristics in bright field observation.

As is apparent from FIG. 9, even if the share amount Δ is made infinitely small, there is no change in the MTF when the contrast is enhanced. Therefore, the lower limit of the share amount Δ can be made as small as possible. Here, when the contrast enhancement method is used, since the differential interference contrast image is captured using the electronic image pickup device 4, in consideration of electrical noise of the electronic image pickup device 4, the higher the intensity of the image to be captured, the better. However, if the share amount Δ is made too small, the intensity of the differential interference image will decrease, as is clear from FIG. From these, the shear amount Δ when the contrast enhancement method is used for the differential interference microscope is 0.08 · λ /
It is preferable that NA ≦ Δ.

In FIG. 1, the shear amount Δ is Δ, where γ is the separation angle between ordinary light and extraordinary light in the Nomarski prism 9 and f ₀ is the focal length of the condenser lens 10.
≈γf ₀ Therefore, it is possible to obtain a desired shear amount Δ by appropriately configuring the Nomarski prism 9 and / or the condenser lens 10.

[0037]

Embodiments of the present invention will be described below.
In the first embodiment of the present invention, in the differential interference microscope shown in FIG. 1, when the wavelength of observation light is λ and the numerical aperture on the object side of the imaging optical system 3 is NA, the share amount Δ is Δ = 0. .20
Configure to λ / NA. Here, assuming that the pupil of the illumination optical system 2 and the pupil of the imaging optical system 3 are in a conjugate relationship with each other through the object plane of the sample 1 to be observed, and their sizes and shapes match each other, the imaging optical system. When the MTF is calculated using the coordinate system in which the radius of the pupil of 3 is standardized to 1, the above formula (10) becomes

[Equation 10] Becomes

FIG. 10 shows Δ = 0.2 in this embodiment.
The MTF characteristic when 0λ / NA is converted into the above coordinate system and calculated by the equation (12). In addition, FIG. 11 shows MT in order to correspond to the contrast enhancement method.
The F characteristic is standardized so that the maximum value becomes 1. For comparison, FIGS. 10 and 11 also show the MTF characteristics in bright-field observation at the same time. Further, Table 1 shows values representing characteristics of the MTF characteristics in FIGS. 10 and 11.

[Table 1]

Here, DIC represents a differential interference microscope,
VEC-DIC represents a differential interference microscope with contrast enhancement. Further, the MTF value of f = 1.64 is focused because f = 1.64 is the frequency corresponding to the Rayleigh resolution.

In the second embodiment of the present invention, in the differential interference microscope shown in FIG. 1, the shear amount Δ is Δ = 0.15λ
/ NA. MTF of differential interference microscope in this case
FIG. 12 shows the characteristics, and FIG. 13 shows the MTF characteristics when the standardization is changed for contrast enhancement. Also, the first
Table 2 shows values representing the characteristics of the respective MTF characteristics, as in the example.

[Table 2]

In the third embodiment of the present invention, in the differential interference microscope shown in FIG. 1, the shear amount Δ is Δ = 0.10λ
/ NA. MTF of differential interference microscope in this case
FIG. 14 shows the characteristics, and FIG. 15 shows the MTF characteristics when the standardization is changed for contrast enhancement. Also, the first
Table 3 shows values representing the characteristics of the respective MTF characteristics, as in the example.

[Table 3]

In the fourth embodiment of the present invention, in the differential interference microscope shown in FIG. 1, the shear amount Δ is Δ = 0.08λ.
/ NA. MTF of differential interference microscope in this case
FIG. 16 shows the characteristics, and FIG. 17 shows the MTF characteristics when the standardization is changed for contrast enhancement. Also, the first
Table 4 shows values representing the characteristics of the respective MTF characteristics, as in the example.

[Table 4]

As described above, four examples have been shown for the shear amount Δ that can obtain a high resolution when the contrast enhancement method is combined with the differential interference microscope, but the present invention is limited to the above four examples. Not the thing, but the share amount Δ
Is Δ <0.25 · λ / NA, preferably 0.08 · λ
If /NA≦Δ<0.25·λ/NA, the same effect as in the above embodiment can be obtained. In particular, in consideration of the electrical S / N characteristics of electronic image pickup devices currently on the market, the shear amount Δ is as shown in the third embodiment:
It is desirable that Δ = 0.1λ / NA.

An example in which the Nomarski prism 9 having the shear amount shown in the third embodiment is manufactured and actually observed will be shown below. The differential interference microscope used for the observation was BX-50 manufactured by Olympus Optical Co., Ltd., and the illumination light was converted into a quasi-monochromatic (λ = 550 nm) by an interference filter, and an oil immersion type PLAPO60xO (NA1.4) was used for the objective lens. A combination of a 2 × intermediate zoom device and a 5 × photographic eyepiece lens is used to project onto an electronic image pickup device at a total magnification of 600 ×. This projection image is received by a television camera C2400-01 manufactured by Hamamatsu Photonics KK as an electronic image pickup device, and the image signal is received by an image processing device XL- manufactured by Olympus Optical Co., Ltd.
An image is displayed on the television monitor C1840-03 manufactured by Hamamatsu Photonics KK via 10. The image displayed on this monitor is a video printer VY-10 manufactured by Hitachi, Ltd.
An image is output with 0.

Using the above-mentioned apparatus, the normal amount and the image obtained by the contrast enhancement method when the shear amount is Δ = 0.1λ / NA shown in the third embodiment of the present invention and the ordinary microscope are used. And an image of a differential interference microscope when a Nomarski prism of 0.38λ / NA is used. First, the results of observing diatoms will be shown. Δ =
The results of ordinary observation at 0.1λ / NA and Δ = 0.38λ / NA without using the contrast enhancement method are shown in FIGS. 18 and 19, respectively. From these results, it can be seen that the contrast decreases as the share amount Δ decreases.

20 and 21 are images obtained by subjecting the images shown in FIGS. 18 and 19 to contrast enhancement processing, respectively. Comparing FIGS. 20 and 21, it can be seen that the resolution is clearly improved in FIG.

Next, images obtained by applying the contrast enhancement method to the observation of the tail villi of the mouse small intestine are shown in FIGS. 22 and 23.
Are shown respectively. Images obtained by observing microtubules extracted from the brain of pigs are shown in FIGS. 24 and 25, respectively. Comparing FIG. 22 and FIG. 23, and FIG. 24 and FIG. 25, respectively,
When the contrast enhancement method is applied, it can be seen that the resolution can be improved in the differential interference microscope by configuring the shear amount Δ to be 0.1λ / NA. 18 to 25 show the image forming magnification of the optical system.
It shows an image with a total magnification of 4700 times multiplied by a monitor magnification and a video printer magnification.

Although an example of observing a biological specimen has been described above, the same effect can be obtained even when the observed specimen is a metal specimen such as a crystal.

Appendix 1. The light from the light source is separated into ordinary light and extraordinary light, which is applied to the observed object, and the image of the observed object is formed by superimposing the ordinary light and the extraordinary light on the imaging plane by the imaging optical system. In the differential interference microscope, the electronic image pickup device arranged on the image forming plane of the image forming optical system and noise components such as illumination unevenness of the optical system are removed from the image signal from the electronic image pickup device, and the noise component is removed. Image processing means for extracting and enhancing an image signal exceeding a preset desired threshold value from the removed image signal, and a separation width Δ in the object plane of the ordinary light and the extraordinary light with which the object to be observed is irradiated. A differential interference microscope, wherein λ is the wavelength of the light to be observed and NA is the numerical aperture on the observation object side of the imaging optical system, and Δ <0.25 · λ / NA is satisfied. . 2. The differential interference microscope according to the above 1, wherein the separation width Δ satisfies 0.08 · λ / NA ≦ Δ.

[0050]

According to the present invention, in the differential interference microscope, the electronic image pickup device is arranged on the image forming surface thereof, and when the image signal from the electronic image pickup device is processed to enhance the contrast, the light to be observed is changed. When the wavelength is λ and the numerical aperture on the observation object side of the imaging optical system is NA, the share amount Δ on the object plane to be observed is configured to be Δ <0.25 · λ / NA. In combination with the contrast enhancement method, it is possible to realize a differential interference microscope with high resolution, and it is possible to easily identify the fine structure of living cells that could not be identified by visual observation.

[Brief description of drawings]

FIG. 1 is a diagram showing a configuration of an example of a differential interference microscope for explaining the principle of the present invention.

FIG. 2 is a diagram showing MTF characteristics in different share amounts.

FIG. 3 is a diagram showing an intensity distribution of an example of an observed object image.

FIG. 4 is a diagram showing an intensity distribution when a contrast enhancement process is performed on the intensity distribution shown in FIG.

FIG. 5 is a diagram showing MTF characteristics when the contrast enhancement of the MTF characteristics shown in FIG. 2 is performed.

FIG. 6 is a diagram showing an MTF characteristic when Δ = 0.5λ / NA.

FIG. 7 is a diagram showing MTF characteristics when Δ = 0.25λ / NA.

FIG. 8 is a diagram showing MTF characteristics in different share amounts.

9 is a diagram showing MTF characteristics when contrast enhancement is performed for each share amount in FIG.

FIG. 10 is a diagram showing an MTF characteristic when the contrast enhancing process is not performed in the first embodiment of the present invention.

FIG. 11 is also M when contrast enhancement processing is performed.
It is a figure which shows TF characteristics.

FIG. 12 is a diagram showing an MTF characteristic when the contrast enhancing process is not performed in the second embodiment of the present invention.

FIG. 13 is also M when contrast enhancement processing is performed.
It is a figure which shows TF characteristics.

FIG. 14 is a diagram showing MTF characteristics in the case where the contrast enhancement processing is not performed in the third embodiment of the present invention.

FIG. 15 is also M when contrast enhancement processing is performed.
It is a figure which shows TF characteristics.

FIG. 16 is a diagram showing MTF characteristics in the case where the contrast enhancement processing is not performed in the fourth embodiment of the present invention.

[FIG. 17] Similarly, M when contrast enhancement processing is performed.
It is a figure which shows TF characteristics.

FIG. 18 is a micrograph of diatom observed by a differential interference microscope according to the present invention in which Δ = 0.1λ / NA.

19 is a photomicrograph of the same specimen as in FIG. 18, observed with a conventional differential interference microscope with Δ = 0.38λ / NA.

20 is a micrograph of the image shown in FIG. 18, which has been subjected to contrast enhancement processing.

FIG. 21 is a micrograph of the image shown in FIG. 19, which has been subjected to contrast enhancement processing.

FIG. 22 is a micrograph obtained by contrast-enhancing an observation image of tail villi of mouse small intestine by a differential interference microscope according to the present invention in which Δ = 0.1λ / NA.

FIG. 23 is a micrograph of a conventional differential interference microscope with Δ = 0.38λ / NA, which is obtained by subjecting an observation image of the same sample as in FIG. 22 to contrast enhancement processing.

FIG. 24 is a micrograph obtained by contrast-enhancing an observation image of microtubules extracted from pig brain by a differential interference microscope according to the present invention in which Δ = 0.1λ / NA.

FIG. 25 is a micrograph of a conventional differential interference microscope with Δ = 0.38λ / NA, which is obtained by subjecting an observation image of the same sample as in FIG. 24 to contrast enhancement processing.

[Explanation of symbols]

 1 Sample 2 Illumination Optical System 3 Imaging Optical System 4 Electronic Imaging Device 5 Image Processing Device 6 Output Device 7 Light Source 8 Polarizer 9, 12 Nomarski Prism 10 Condenser Lens 11 Objective Lens 13 Analyzer

Claims (2)

[Claims] 1. The light from a light source is separated into ordinary light and extraordinary light to irradiate an object to be observed, and an image of the object to be observed is superposed on the image plane by the ordinary light and the extraordinary light. In a differential interference microscope configured to form an image, an electronic image pickup device is arranged on an image forming surface of the image forming optical system, and a separation width Δ in the object plane of the ordinary light and the extraordinary light with which the observed object is irradiated, When the wavelength of the light to be observed is λ and the numerical aperture of the imaging optical system on the observation object side is NA, Δ
A differential interference microscope configured to satisfy <0.25 · λ / NA so as to process an image signal from the electronic image pickup device to enhance contrast. 2. The differential interference microscope according to claim 1, wherein the separation width Δ satisfies 0.08 · λ / NA ≦ Δ.