EP2893385A1 - Confocal laser scanning microscope with a laser light source triggered in a pulsed manner - Google Patents
Confocal laser scanning microscope with a laser light source triggered in a pulsed mannerInfo
- Publication number
- EP2893385A1 EP2893385A1 EP13756500.8A EP13756500A EP2893385A1 EP 2893385 A1 EP2893385 A1 EP 2893385A1 EP 13756500 A EP13756500 A EP 13756500A EP 2893385 A1 EP2893385 A1 EP 2893385A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- scanning microscope
- laser scanning
- input
- signal
- pulse
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
-
- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0096—Microscopes with photometer devices
Definitions
- the present invention relates to a confocal laser scanning microscope with a laser light source and a Ver ⁇ drive for adjusting the intensity of a laser light source in a confocal laser scanning microscope.
- the present invention is in the field of konfoka ⁇ len laser scanning microscopy (CLSM engl. "Confocal scanning laser microscopy”), such as in the US 7,477,449 B2 described.
- CLSM engl. "Confocal scanning laser microscopy” For imaging, a spot on the sample to be observed is illuminated here with a focused laser beam, and the reflected or fluorescent light emanating therefrom is detected by a sensor which is likewise focused on this location, as a result of which a high resolution can be achieved.
- the intensity should be regulated as closely as possible and thereby cover a large Intensticiansbe ⁇ rich, so that it can be for as many different fluorescent dyes or fluorochromes is ⁇ sets.
- a modulation frequency down to the fre- range of one MHz is sought.
- the minimum adjustable intensity should be as close to zero as possible.
- a confocal laser scanning microscope is equipped with a pulsed controlled laser light source, preferably a laser diode.
- the pulsed control provides more degrees of freedom, of which at least one pulse amplitude and pulse width ⁇ be used in the present invention.
- the invention improves the Rege ⁇ lung Q and the dynamics and increases the available brightness range, in particular to small light intensities, as well as the wavelength stability.
- the invention has particular relation to a continu ⁇ ous control special advantages.
- a modulation frequency ⁇ up in the frequency range of one MHz into or higher is possible.
- the minimum adjustable intensity is close to zero. When switching on and off, there are hardly any overshoots or undershoots of the power output. Furthermore, the signal-to-noise ratio is improved, in particular for low intensities.
- the invention enables a broad spectrum for the control.
- the amount of light emitted to the sample is the integral of the intensity over the duration of the irradiation.
- the control circuit is so arranged that the laser light source intensity with a drive signal adjustable in ⁇ and is supplied period of time.
- the duration of the current can be set below 100 ps. A maximum time is possible until continuous operation.
- the time ⁇ duration of the current pulse may be variable, and preferably also externally substantially continuously (or in steps smallest digital control) can be set.
- a lower amplitude threshold value (so-called laser threshold or threshold current intensity) is specified below which a safe emission of laser light is not guaranteed.
- the on ⁇ control circuit is preferably set up so that the ⁇ ser is not exceeded . For a further reduction of the emitted light power at the lasing threshold is the duty cycle or the pulse ⁇ wide as a degree of freedom.
- an at least two-dimensional map of pulse amplitude and pulse width can be spanned, wherein each pair of values characterizes a mean light intensity or quantity of light.
- a vector or pair of values of the pulse amplitude and pulse width can be found at least ⁇ ge.
- a plurality of suitable pairs of values will be available in the map, so that other boundary conditions, such as an intensity noise, a certain line receiving the system, a maximum effi ⁇ enz of excitation, minimal fading of the sample, a special suitability for a certain fluorescent dye, etc., can be taken into account.
- the detection of measuring light is adapted to the time course (pulse width and / or frequency) of the drive signal.
- the on ⁇ control circuit therefore has an input and / or output (eg. TTL or switching contact) for a pulse frequency of the drive signal on.
- the pulse frequency can be predetermined by ex ⁇ tern, for example of a measuring device, or the pulse frequency can be specified externally, for example, to the measurement device.
- a tuning of an excitation frequency with a detection or sampling frequency is possible, as a result of which, in particular moire artifacts in the detection can be avoided.
- FIG. 1 shows schematically a beam path in a confocal laser scanning microscope according to a ⁇ preferred embodiment of the invention.
- FIG. 2 schematically shows a preferred embodiment of a lighting device of a confocal laser scanning microscope according to the invention. Detailed description of the drawing
- FIGs 1 and 2 are described together, wherein schematically illustrates a preferred embodiment of a erfindungsge ⁇ MAESSEN confocal laser scanning microscope in figure 1 ones shown, and indicated generally at 100 and schematic manner in Figure 2, the preferred in the invention Be ⁇ illumination unit 1 is shown in more detail.
- the microscope has a designated 1communsein ⁇ direction having a recess formed as a laser diode laser light source ⁇ 41st
- the laser light source 41 emits an illumination light beam 5, which passes through a beam splitter 21 and an objective 23 and strikes a sample 25.
- Measurement light 27 reflected by the specimen or generated by fluorescence enters through the objective 23 and is decoupled at the beam splitter 21 into an observation beam path and fed to a detector 51.
- a drive circuit 40 which supplies the laser diode 41 with power.
- the drive circuit 40 is to ⁇ directed to receive a set amount of light as a setpoint and to process.
- the setpoint is supplied in the present example by a computing unit 39 which is adapted to STEU ⁇ augmentation of the microscope 100th Furthermore, measurement data are transmitted from the detector 51 to the arithmetic unit 39 and evaluated there.
- the arithmetic unit 39 also controls the detector 51.
- the drive circuit 40 has a plurality of functional units, which will be explained below.
- a scarf ⁇ processing part 44 is formed as a control circuit part, eg ASIC or FPGA, and designed to control the drive circuit 40 and set up.
- the control circuit ⁇ part 44 has an input for an input variable S and a terminal I / A for a frequency signal, which are connected to the arithmetic unit 39 in the embodiment shown.
- a setpoint signal is input output quantity S, here a target intensity or target amount of light supplied.
- the port I / O may serve to improve the operation of the detector 51, as explained later.
- the control circuit part 44 is configured to determine from ei ⁇ ner supplied target amount of light based on a particular stored in the control circuit part 44 map 45 each have a setpoint for a duty cycle R (ratio of pulse width W to pulse period T) and a pulse amplitude A.
- the duty cycle R is supplied to a PWM circuit part 46 which generates a PWM signal therefrom.
- the target value for the pulse amplitude A is a PAM 47 supplied to circuit section together with the generated from the PWM circuit portion 46 PWM signal, from which the PAM circuit part 47 a in the invention with variable pulse width W and pulse duty factor R and variable Pul ⁇ samplitude A generated drive signal 48 generated, which is guided via a current measuring circuit part 49 to the laser diode 41.
- the current measuring circuit part 49 is used as part of a laser control implemented according to the preferred embodiment shown here.
- the illumination device 1 for a return or measurement of the illumination light 5 in the context of laser control on a photodiode 43 and a photometering circuit 42.
- Pho ⁇ todiode 43 is a small part of the illumination light 5 decoupled.
- the control circuit part 44 can regulate the generation of the drive signal 48 via the returned current and intensity or light quantity values.
- the invention enables to specifically adjust pulse amplitude and pulse width for each pulse of the drive signal 48 and thus to specify an average light intensity and light amount of the Be ⁇ leuchtungsstrahls. 5 Within the scope of the invention, very small average intensities and amounts of light can be achieved, in particular by reducing the pulse width.
- the control circuit 40 is connected to the arithmetic unit 39 via the terminal I / O and transmits via the terminal I / O as output a frequency signal indicating the frequency of the drive ⁇ signais 48 describes.
- This allows the arithmetic unit 39, the measurement with the detector 51 to carry out with a sampling frequency which depends on the frequency of the driving signal 48 in order to avoid in this way, for example Moire artifacts of Mes ⁇ solution.
- the port I / O can serve as an input, or one port each can be provided as an output and as an input.
- the frequency of the drive signal 48 would be predefined as a function of the sampling frequency. In both cases, is advantageously achieved that the frequency of the drive signal the Fre ⁇ 48 is an integer multiple (1, 2, 3, ...) is the sampling frequency.
Landscapes
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Microscoopes, Condenser (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
Description
Claims
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102012215932 | 2012-09-07 | ||
DE102012218624.7A DE102012218624B4 (en) | 2012-09-07 | 2012-10-12 | Confocal laser scanning microscope with a pulsed laser light source |
PCT/EP2013/068286 WO2014037401A1 (en) | 2012-09-07 | 2013-09-04 | Confocal laser scanning microscope with a laser light source triggered in a pulsed manner |
Publications (1)
Publication Number | Publication Date |
---|---|
EP2893385A1 true EP2893385A1 (en) | 2015-07-15 |
Family
ID=50153343
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP13756500.8A Withdrawn EP2893385A1 (en) | 2012-09-07 | 2013-09-04 | Confocal laser scanning microscope with a laser light source triggered in a pulsed manner |
Country Status (5)
Country | Link |
---|---|
US (1) | US9575300B2 (en) |
EP (1) | EP2893385A1 (en) |
JP (1) | JP6389462B2 (en) |
DE (1) | DE102012218624B4 (en) |
WO (1) | WO2014037401A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6501112B2 (en) * | 2015-03-25 | 2019-04-17 | 国立大学法人山梨大学 | Cloud particle observation microscope |
EP3159676B1 (en) * | 2015-10-23 | 2018-04-04 | Abberior Instruments GmbH | Method and device for high precision imaging of a structure of a sample marked with fluorescence markers |
WO2017195256A1 (en) * | 2016-05-09 | 2017-11-16 | オリンパス株式会社 | Optical scanning type observation device and optical scanning type observation method |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110296562A1 (en) * | 2009-02-04 | 2011-12-01 | Ecole Polytechnique | Method and device for acquiring signals in laser scanning microscopy |
Family Cites Families (16)
Publication number | Priority date | Publication date | Assignee | Title |
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US6745067B1 (en) | 1998-09-14 | 2004-06-01 | Lucid, Inc. | System for marking the locations of imaged tissue with respect to the surface of the tissue |
US6525862B2 (en) * | 1996-10-30 | 2003-02-25 | Photogen, Inc. | Methods and apparatus for optical imaging |
EP1108227B1 (en) * | 1998-11-16 | 2008-01-02 | Leica Microsystems CMS GmbH | Method for operating a confocal laser scanning microscope |
DE19919091C2 (en) | 1999-04-27 | 2002-01-17 | Zeiss Carl Jena Gmbh | Arrangement for setting the laser power and / or the pulse length of a short-pulse laser in a microscope |
EP1102101A1 (en) * | 1999-11-22 | 2001-05-23 | Leica Microsystems Heidelberg GmbH | Laser scanning microscope |
AU2002357016A1 (en) * | 2001-11-28 | 2003-06-10 | James W. Overbeck | Scanning microscopy, fluorescence detection, and laser beam positioning |
DE10253108B4 (en) | 2002-11-13 | 2005-11-03 | Leica Microsystems Heidelberg Gmbh | Method for detecting an object with a scanning microscope and a scanning microscope for detecting an object |
DE10253609A1 (en) | 2002-11-15 | 2004-05-27 | Leica Microsystems Heidelberg Gmbh | scanning microscope |
DE10332060A1 (en) | 2003-07-11 | 2005-02-03 | Carl Zeiss Jena Gmbh | Method for operating a laser scanning microscope |
US7253950B2 (en) * | 2003-07-17 | 2007-08-07 | Olympus Corporation | Scanning laser microscope |
US9055867B2 (en) * | 2005-05-12 | 2015-06-16 | Caliber Imaging & Diagnostics, Inc. | Confocal scanning microscope having optical and scanning systems which provide a handheld imaging head |
DE102005047884A1 (en) * | 2005-10-06 | 2007-04-26 | Leica Microsystems Cms Gmbh | Scanning microscope and scanning method with a scanning microscope |
DE102007002203A1 (en) * | 2007-01-16 | 2008-07-17 | Carl Zeiss Microimaging Gmbh | Lighting device and lighting method |
DE102009055993B4 (en) * | 2009-11-26 | 2016-06-02 | Leica Microsystems Cms Gmbh | A method of inspecting an object with the aid of a microscope and a microscope-like device for examining an object |
JP5544231B2 (en) | 2010-07-15 | 2014-07-09 | 富士フイルム株式会社 | Endoscope light source device and endoscope system |
JP5481294B2 (en) * | 2010-07-15 | 2014-04-23 | 富士フイルム株式会社 | Endoscope system |
-
2012
- 2012-10-12 DE DE102012218624.7A patent/DE102012218624B4/en active Active
-
2013
- 2013-09-04 WO PCT/EP2013/068286 patent/WO2014037401A1/en active Application Filing
- 2013-09-04 JP JP2015530377A patent/JP6389462B2/en active Active
- 2013-09-04 EP EP13756500.8A patent/EP2893385A1/en not_active Withdrawn
- 2013-09-04 US US14/426,260 patent/US9575300B2/en active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110296562A1 (en) * | 2009-02-04 | 2011-12-01 | Ecole Polytechnique | Method and device for acquiring signals in laser scanning microscopy |
Non-Patent Citations (4)
Title |
---|
ANONYMOUS: "Demonstration of the confocal microscope Leica TCS SP8 - Cell Biology Utrecht University", 21 August 2012 (2012-08-21), XP055575947, Retrieved from the Internet <URL:https://cellbiology.science.uu.nl/facilities/biology-imaging-center-bic/activities/demonstration-of-the-confocal-microscope-leica-tcs-sp8/> [retrieved on 20190401] * |
ANONYMOUS: "Leica claims fastest true confocal scanner - LabHomepage", 25 September 2012 (2012-09-25), XP055575951, Retrieved from the Internet <URL:http://labhomepage.com/2651/microscope/leica-claims-fastest-true-confocal-scanner/> [retrieved on 20190401] * |
LEICA MICROSYSTEMS: "Living up to Life User Manual Leica TCS SP8 SMD for FCS, FLIM and FLCS", 30 May 2012 (2012-05-30), XP055575940, Retrieved from the Internet <URL:https://www.well.ox.ac.uk/files-library/lecia-sp8-confocal-user-manual.pdf/@@download> [retrieved on 20190401] * |
See also references of WO2014037401A1 * |
Also Published As
Publication number | Publication date |
---|---|
JP6389462B2 (en) | 2018-09-12 |
DE102012218624A1 (en) | 2014-03-13 |
WO2014037401A1 (en) | 2014-03-13 |
JP2015530616A (en) | 2015-10-15 |
US9575300B2 (en) | 2017-02-21 |
DE102012218624B4 (en) | 2020-07-09 |
US20150212306A1 (en) | 2015-07-30 |
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