US008492693B2 (12) United States Patent (10) Patent No.: Brooker (54) (75) (45) Date of Patent: AUTOFOCUS APPARATUS Inventor: US 8,492,693 B2 Jeffrey S. Brooker, Oak Hill, VA (US) Jul. 23, 2013 2004/0135061 A1 7/2004 Kreh 2005/0121596 A1 6/2005 Kam et al. 2009/0021724 A1 * 1/2009 Mahadevan'lansen et al. (73) Assignee: Thorlabs, Inc., Newton, NJ (US) (*) Notice: ............................. .. 356/73 FOREIGN PATENT DOCUMENTS Subject to any disclaimer, the term of this patent is extended or adjusted under 35 W0 Zoos/029506 3/2008 OTHER PUBLICATIONS U.S.C. 154(b) by 313 days. (21) International Search Report and Written Opinion for corresponding International Application No. PCT/US20l0/05298l mailed Jan. 20, Appl, NO; 12/906,086 201 l. (22) Filed: Oct. 16, 2010 * cited by examiner (65) Prior Publication Data US 2011/0090562 A1 Apr. 21, 2011 Primary Examiner * Seung C Sohn (74) Attorney, Agent, or Firm * Graham Curtin, P.A. Related US. Application Data (60) Provisional application No. 61/252,263, ?led on Oct. (57) 16’ 2009' An autofocus apparatus includes, in one embodiment, a light (51) Int. Cl. G02B 7/04 G02B 27/64 (52) source; a s P litter; a ?ber o P tic circulator; an o P tical collima tor; a balance detector; and a microprocessor. The ?ber optic circulator couples one of the split light signals at a ?rst port, to the optical collimator at a second port, and to the balance (2006 01) (200601) (200601) G02B 27/40 U 5 Cl ' detector at the third port. The optical collimator directs the U'SI;C ' 58 250/201 4 F 1d "" "_ "" ( ) Ulsepco S """ """""""" " through a Dichroic mirror and a microscope objective. The assl “ugh/263:2 214 1 214 R_ 356/73 balance detector uses another one of the split light signals as ' ’ 1, 1 _ ?l f ee app lcanon ' ’ ’356/479’ h h, e or Comp ete Seam an input, and converts a light signal, re?ected off of a sub strate the sample is placed on, into an analog voltage signal. lstory' The microprocessor processes the output of the balance References Cited detector and position feedbacks from an adjustable micros copy stage to generate a command for moving the position of the adjustable microscopy stage to achieve a desired focus. U.S. PATENT DOCUMENTS 5,654,799 A 6,900,943 B2 light beam from the ?ber optic circulator onto a sample ' """""""" " (56) ABSTRACT 8/1997 Chase et a1. 5/2005 Andersen et al. 200 11 Claims, 3 Drawing Sheets LIGHT SOURCE K 110 2 Axis Stage with position feedback 240\ PD Detector Optical Output Voltage Output Microprocessor A/D converter Position Feedback ZAxis drive 195 198 US. Patent Jul. 23, 2013 Sheet 1 of3 US 8,492,693 B2 LIGHT SOURCE 100 122 Z T Splitter 110 xi 8 ‘I24 195 with sol/2min izgjback ] 98 1 Objective 1 50 A 1 60 —- 130 IV Dichroic _ COllimator '1 mlrror 1 Circulator I |ir\|n(;r3i1rag| 140 3 i 70 Path \ Balance Detector RF Output) Microprocessor /.-'| Position Converter Feedback 0 utput FIG. I I I ZAXIS drlVe US. Patent Jul. 23, 2013 2001 Sheet 2 of3 US 8,492,693 B2 LIGHT SOURCE Z Axis Stage with position feedback 240\ PD Detector Optical Output Voltage Output D Microprocessor /—'| €AID Position converter Feedback Output FIG. 2 ZA ' d ' X's We K- I US. Patent Jul. 23, 2013 Sheet 3 of3 Signal Voltage US 8,492,693 B2 / /\ 1/ ,/ \J Stage Position FIG. 3 \ US 8,492,693 B2 1 2 AUTOFOCUS APPARATUS uses the ?rst portion of the light signal as an input; and a microprocessor for processing the analog voltage signal from CROSS-REFERENCE TO RELATED APPLICATIONS the balance detector. In another embodiment of the present invention, an auto This application claims the bene?t of US. Provisional Application No. 61/252,263 ?led on Oct. 16, 2009, the con tents of Which are herein incorporated by reference. includes: a light source; an ?ber optic circulator having a ?rst focus microscope apparatus is provided. The apparatus port, second port and a third port; Wherein the light signal in the ?ber optic circulator travels only from the ?rst port to the second port and from the second port to the third port; Wherein the ?ber optic circulator couples to a light signal FIELD OF THE INVENTION output of the light source at the ?rst port; an optical collimator for directing a light output from the second port of the ?ber optic circulator onto a sample through a Dichroic mirror and a microscope objective, Wherein the sample is placed on an adjustable microscopy stage; a photodiode detector for con verting a light signal, re?ected off of a substrate that the The invention generally relates to a microscopy apparatus, and more particularly to techniques for automatically adjust ing the position of a stage for attaining proper focus. BACKGROUND sample is placed on, into an analog voltage signal, Wherein the re?ected light signal is captured by the microscope objec As With all optical systems, microscopes suffer from diminished depth of ?eld as the magni?cation and the NA (numerical aperture) of the imaging lens (objective) 20 increases. When using a microscope, the user is responsible tive and sent to the photodiode detector through the Dichroic mirror, the optical collimator, and into the second port and out of the third port of the ?ber optic circulator; and a micropro for attaining proper focus of the sample by moving the sample cessor for processing the analog voltage signal from the pho relative to the objective. When microscopy is automated and todiode detector. In yet another embodiment of the present invention, a the user is no longer involved in looking at each image, a method of auto focusing is required. In the related art, tech 25 method for operating a microscopy apparatus is provided. niques that achieve automatic focus by gauging the distance The method includes the steps: illuminating a light beam to a betWeen the front lens and the bottom of the container (e. g., slide, Well plate, etc.) are described. Such techniques are based on re?ecting a beam of light off of the ?rst surface and splitter for splitting the light beam into a ?rst portion of a light signal and a second portion of a light signal; inputting the ?rst portion of the light signal to a balance detector; coupling an ?ber optic circulator to the second portion of the light signal measuring the re?ection. The de?ciency of such techniques, 30 hoWever, is that if the container that the sample is on has an at a ?rst port, to an optical collimator at a second port, and to inconsistent thickness, as in most plastics, then the resulting 35 a balance detector at the third port, Wherein the light signal in the ?ber optic circulator travels only from the ?rst port to the second port and from the second port to the third port; direct ing a light beam from the second port of the ?ber optic circulator onto a sample by the light collimator through a Dichroic mirror and a microscope objective, Wherein the sample is placed on an adjustable microscopy stage; captur 40 tive and sending to the balance detector through the optical image can be off in focus the amount of the deviation of the substrate. Cellular imaging relies on the groWth of cells on the bottom of a glass or plastic substrate. The cells groW parallel to the surface and secrete proteins that cause them to adhere to the substrate. In order to maintain the groWth of the cells, nutrient rich liquid medium is added to feed the cells and maintain proper physiological conditions. In this scenario, the surface ing the re?ected light beam signal by the microscope obj ec collimator and into the second port and out of the third port of of the plastic is covered in an aqueous solution, Which can be used to detect the position of the cells. The index of refraction the ?ber optic circulator; converting the light signal re?ected change betWeen the plastic and the liquid can be located using a loW noise, high sensitivity re?ected light setup. voltage signal by the balance detector; and processing the off of a substrate that the sample is placed on, into an analog 45 SUMMARY output of the balance detector. The present invention may be realiZed as an autofocus microscopy apparatus in one embodiment. The microproces In an embodiment of the present invention, an autofocus sor may generate a command for moving the position of the microscope apparatus is provided. The apparatus includes: a light source; a splitter for splitting a light beam illuminated by adjustable microscopy stage to achieve a desired optical focus, based on the output of the balance detector and position 50 the light source to a ?rst portion of a light signal and a second feedbacks from the adjustable microscopy stage. portion of a light signal; an ?ber optic circulator having a ?rst port, second port and a third port; Wherein the light signal in the ?ber optic circulator travels only from the ?rst port to the second port and from the second port to the third port; Wherein the ?ber optic circulator couples to the second por tion ofthe light signal at the ?rst port; an optical collimator for directing a light output from the second port of the ?ber optic In another embodiment, the present invention may be real iZed as a tracking device. The microprocessor may continu ously monitor the analog voltage signal and generate a com 55 circulator onto a sample through a Dichroic mirror and a microscope objective, Wherein the sample is placed on an adjustable microscopy stage; a balance detector for convert ing a light signal, re?ected off of a substrate that the sample is placed on, into an analog voltage signal, Wherein the re?ected 60 voltage signal and seeks to change the distance betWeen the sample and the microscope objective to maximize the voltage from the detector, or maintain the voltage at a speci?ed point. In another embodiment, the microprocessor may deter mine the position of the sample based on the analog voltage signal and position feedbacks. light signal is captured by the microscope objective and sent to the balance detector through the Dichroic mirror, the opti cal collimator, and into the second port and out of the third port of the ?ber optic circulator; Wherein the balance detector mand in order to maintain a speci?c distance from the sample, or Wherein the microprocessor continuously monitors the BRIEF DESCRIPTION OF THE DRAWINGS 65 FIG. 1 is a diagram of an autofocus apparatus designed in accordance With an embodiment of the invention. US 8,492,693 B2 3 4 FIG. 2 is a diagram of an autofocus apparatus designed in accordance With another embodiment of the invention. FIG. 3 is a plot of signal voltage as a function of stage signal from the objective 160 into an analog voltage signal that is then converted to a digital signal by the A/ D converter in the microprocessor 180. The microprocessor 180, based on the value of the digital position. signal and position feedbacks, moves the microscopy stage along the Z axis by commanding the controller 190. As shoWn in FIG. 1 for example, the sample 195 is placed on the micros copy stage 198 having an adjustable position along the DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS This disclosure describes the best mode or modes of prac Z-axis, thereby alloWing adjustment of the separation ticing the invention as presently contemplated. This descrip betWeen the sample 195 and objective 160 to maintain focus. The microprocessor 180 also records the voltage values mea sured by the balance detector 140 as Well as the position of the adjustable microscopy stage at different distances betWeen tion is not intended to be understood in a limiting sense, but provides an example of the invention presented solely for illustrative purposes by reference to the accompanying draW ings to advise one of ordinary skill in the art of the advantages and construction of the invention. In the various vieWs of the draWings, like reference characters designate like or similar parts. It is to be noted that all ?ber optic systems can be the sample 195 and the objective 160. The voltage signal peaks are analyZed and then the microscopy stage 198 is commanded to move, by the controller 190, to the position of the voltage signal peak that best correlates to the desired optical focus. FIG. 3 shoWs an example plot of the voltage replaced With free space equivalents. FIG. 1 shoWs an exemplary and non-limiting diagram of an autofocus apparatus 100 designed in accordance With an embodiment of the invention. The apparatus 100 includes at least a light source 110, a ?ber optic splitter 120, a balance detector 140, a ?ber optic circulator 130, an optical collimator 150, a microprocessor 180, and a controller 190. The light source 110 could include, but not be limited to, a laser diode, for example. Other light sources are contemplated. 20 25 In accordance With principles of the invention, light illu minated by the light source 110 is ?ber coupled or focused into the ?ber optic splitter 120 Which directs a ?rst portion 122 of the original light into one of the ports of the balance 30 FIG. 2 shoWs an exemplary diagram of an autofocus appa ratus 200 implemented in accordance With another non-lim iting embodiment of the invention. In this embodiment the apparatus 200 does not include a ?ber optic splitter and a simple photodiode detector 240 is used instead of a balance detector. Light illuminated by the light source 110 is ?ber coupled or focused into the ?rst port (1) of the ?ber optic circulator 130. The light travels in the circulator 130 and outputs at the second port (2), Where the light signal is focused into a beam by the collimator 150. Light signal obtained back from the collimator 150 is input into the circu lator 130 at the second port (2) and is output at the third port (3). The output from the circulator 130 is fed into the photo diode detector 240. The photodiode detector 240 converts the detector 140. In one embodiment of the invention the Wave length of the light source 110 is a 1310 nm. Other values are contemplated. The ?ber optic circulator 130 comprises a ?rst port (1), a second port (2) and a third port (3). The second portion 124 output from the splitter 120 is fed into the ?rst port (1) of the ?ber optic circulator 130. The second portion 124 is signi?cantly greater than the ?rst portion 122 (for example, 99% versus 1%). The light travels in the circulator 130 and outputs at the second port (2), Where the light signal signal peaks against the position of the microscopy stage. 35 light signal into a voltage signal output. The implementation illustrated in FIG. 2 is e?icient When a time course of the change in the light intensity is longer than the period of time 40 for the movement of the stage. It should be appreciated by one of ordinary skill in the art that the autofocus mechanisms illustrated in FIGS. 1 and 2 are designed as a loW background high sensitivity detector using is collimated into a beam by the collimator 150. It should be noted that a light signal can travel in the circulator 130 only a ?ber optic circulator. The addition of the circulator to the from the ?rst port (1) to the second port (2) and to third port the sample from being visible to the detector. This means of (3). light path prevents the light that is being sent from the laser to 45 designed to only re?ect Wavelengths above a prede?ned Wavelength and pass anything beloW. In an exemplary embodiment the prede?ned Wavelength is 1300 nm. The light beam travels through the microscope objective 160 and is The autofocus system of the present invention is particu larly suited for high content screening and biological imag 50 copy stage. The light re?ects off of the plastic or glass sub strate that the sample 195 is placed on, is captured by the objective 160, and then sent back into the ?ber by the colli than With current solutions. Some of the cell-based as says that 55 balance detector 140 circuitry the signal from the objective 160 is electronically divided by the signal from the ?rst por tion 122 of the original light provided by the splitter 120, thus cancelling out any change in the light source intensity over time. The balance detector 140 further converts the corrected are supported include, but are not limited to, nuclear-cyto plasmic translocation, plasma membrane translocation, pro tein expression, lipid droplet formation, DNA content, cell toxicity, cell viability (apoptosis/necrosis) and others. The light signal from the collimator 150 is input into the circulator 130 at the second port (2) and is output at the third port (3), as the light travels only in one direction through the circulator 130. The output from the circulator 130 is fed into another one of the ports of the balance detector 140. In the ing. In one embodiment, the autofocus could be used With an automated ?uorescence microplate imaging system that is designed to enable investigators to develop cell-based assays faster, at higher spatial resolution, and more economically focused on the sample 195 placed on an adjustable micros mator 150. If the sample 195 is not contained in a glass or plastic substrate, the re?ection can come from the sample 195 itself. illumination reduces the background signal to the detector, by the amount of blocking that the circulator provides. In one embodiment the blocking is better than 45 db. The beam re?ects off of a Dichroic mirror 170, Which is As shoWn in FIG. 1, position feedback is facilitated using, 60 in one embodiment, linear encoder scales embedded Within the stage construction. While linear encoder scales are described herein, other positioning means are contemplated. One non-limiting example is for microscopy Where the stage is used to either position any part of a microscope With 65 respect to a sample, or a sample With respect to a microscope. One example of this application is shoWn in connection With FIGS. 1-2 herein. Another non-limiting example includes US 8,492,693 B2 6 5 a microprocessor for processing the analog voltage signal machine vision inspection, or non contact-based dimensional inspection. Other industries and applications are contem from the photodiode detector. plated. 2. The apparatus of claim 1, Wherein a time course of the It is contemplated that the position of the sample may change in the light intensity of the light source is longer than change over time. It may be desirable to keep track of the position of the sample in order to maintain focus. In an embodiment of the present invention, the system can be real iZed as a tracking device. Using the principles of the present invention, the tracking device can maintain a speci?c distance the period of time for the movement of the adjustable micros copy stage. 3. The apparatus of claim 1, Wherein the microprocessor generates a command for moving the position of the adjust able microscopy stage to achieve a desired optical focus betWeen the device and the sample by tracking the signal voltage and continuously adjusting the Z-position. When the position of the sample 195 changes, the re?ected light signal based on voltage signal peaks measured by the photodiode detector. 4. The apparatus of claim 1, Wherein the microprocessor continuously monitors the voltage signal and generates a command for moving the position of the adjustable micros received by the balance detector 140 or the photodiode detec tor 240 changes. As a result, microprocessor 180 detects a change in the voltage signal from the balance detector 140 or the photodiode detector 240. Microprocessor 180 generates a corresponding movement command in order to receive the same voltage from the detector, and thus maintains the same distance betWeen the objective 160 and the sample 195. The present invention can be used as a metrology tool. copy stage in order to maintain a speci?c distance betWeen the sample and the microscope objective. 20 maintain a speci?c distance betWeen the sample and the Speci?cally, the invention provides a good non-contact tech nique for measurement and distance gauging. In another embodiment of the present invention, the system can be realiZed as a distance measuring device. In this case, there is no need to issue any movement command. The microscope objective. 6. The apparatus of claim 1, Wherein the microprocessor determines the position of the sample based on the analog 25 re?ected light signal received by the balance detector 140 or the photodiode detector 240 is used to generate an analog voltage signal.As shoWn in FIG. 3, the voltage depends on the position of the stage. Microprocessor 180 analyZes the volt age signal and position feedbacks from the stage and thus determines the position of the sample. 7. A method for automatically operating an autofocus coupling an ?ber optic circulator to a light signal output of 30 second port, and to a photodiode detector at the third 35 40 foreseen, may nonetheless represent equivalents thereto. What is claimed is: 45 1. An autofocus apparatus, comprising: a light source; an ?ber optic circulator having a ?rst port, second port and a third port; Wherein the light signal in the ?ber optic circulator travels only from the ?rst port to the second port and from the second port to the third port; Wherein into an analog voltage signal, Wherein the re?ected light signal is captured by the microscope objective and sent to the photodiode detector through the Dichroic mirror, the optical collimator, and into the secondport and out of the third port of the ?ber optic circulator; and travels only from the ?rst port to the second port and from the second port to the third port; directing a light beam from the second port of the ?ber optic circulator onto a sample by the light collimator through a Dichroic mirror and a microscope objective, Wherein the sample is placed on an adjustable micros copy stage; capturing the re?ected light beam signal by the microscope objective and sending to the photodiode detector through the optical collimator and into the second port and out of the third port of the ?ber optic circulator; converting the light signal re?ected off of a substrate that the sample is placed on, into an analog voltage signal by the photodiode detector; and processing the output of the photodiode detector. 8. The method of claim 7, Wherein a time course of the 50 the ?ber optic circulator couples to a light signal output of the light source at the ?rst port; an optical collimator for directing a light output from the second port of the ?ber optic circulator onto a sample through a Dichroic mirror and a microscope objective, Wherein the sample is placed on an adjustable micros copy stage; a photodiode detector for converting a light signal, re?ected off of a substrate that the sample is placed on, a light source at a ?rst port, to an optical collimator at a port, Wherein the light signal in the ?ber optic circulator ticular embodiment, but it is to be construed With references to the appended claims so as to provide the broadest possible interpretation of such claims in vieW of the prior art and, therefore, to effectively encompass the intended scope of the invention. Furthermore, the foregoing describes the invention in terms of embodiments foreseen by the inventor for Which an enabling description Was available, notWithstanding that insubstantial modi?cations of the invention, not presently voltage signal from the photodiode detector and position feedbacks received from the adjustable microscopy stage. apparatus, the method comprising: While the present invention has been described at some length and With some particularity With respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any par 5. The apparatus of claim 1, Wherein the microprocessor continuously monitors the voltage signal and generates a command for moving the position of the objective in order to change in the light intensity of the light source is longer than the period of time for the movement of the adjustable micros copy stage. 9. The method of claim 7, further comprising generating a command for moving the position of the adjustable micros 55 copy stage to achieve a desired optical focus based on voltage signal peaks measured by the photodiode detector. 10. The method of claim 7, further comprising continu ously monitoring the voltage signal and generating a com mand for moving the position of the adjustable microscopy 60 stage in order to maintain a speci?c distance betWeen the sample and the microscope objective. 1 1. The method of claim 7, further comprising determining the position of the sample based on the analog voltage signal from the photodiode detector. * * * * *
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