# US 8,570,649 B2 · Dual-mode raster point scanning/light sheet illumination microscope

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- Number: US 8,570,649 B2
- Document ID: US8570649B2
- Status: Granted
- Kind: B2
- Application number: 12/916,124
- Filed: 2010-10-29
- Published: 2011-06-09
- Granted: 2013-10-29
- Earliest claimed: 2009-10-29
- Assignees: California Institute of Technology
- Applicants: Thai V. Truong; John M. Choi; Scott E. Fraser; Willy Supatto; David S. Koos
- Inventors: Thai V. Truong; John M. Choi; Scott E. Fraser; Willy Supatto; David S. Koos
- Web page: https://patentgrep.com/patent/US8570649B2

## CPC

- G02B 21/16: adapted for ultraviolet illumination {; Fluorescence microscopes (G02B21/0076 takes precedence)}
- G01N 21/6408: {with measurement of decay time, time resolved fluorescence}
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6458: {Fluorescence microscopy (fluorescence microscopes per se G02B21/0076 and G02B21/16)}
- G01N 21/6486: {Measuring fluorescence of biological material, e.g. DNA, RNA, cells (G01N21/6428 takes precedence)}
- G02B 21/002: {Scanning microscopes (scanning near field optical microscopes G01Q60/18)}
- G02B 21/367: {providing an output produced by processing a plurality of individual source images, e.g. image tiling, montage, composite images, depth sectioning, image comparison}
- G01N 2021/655: {Stimulated Raman}
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

An apparatus for and method of performing light sheet microscopy (LISH) and light scanning microscopy (RAPS) in a single device are provided. The dual-mode imaging microscope allows for the use of both LISH and RAPS in a single instrument. This dual-mode device will allow researchers to have access to both types of microscopy, allowing access to the widest possible selection of samples. In addition, the device will reduce the high costs and space requirements associated with owning two different microscopes (LISH and RAPS).

## Claims

### Claim 1

A dual-mode imaging microscope comprising:
an excitation source, said excitation source being capable of producing an excitation beam, and being disposed such that the excitation beam is directed along at least one excitation beam path, wherein the at least one excitation beam path is provided with a scanning optic capable of scanning said excitation beam in at least two dimensions;
a sample holder in optical communication with said excitation source such that a sample contained therein is excited by said excitation beam;
a light scanning microscope unit comprising:
a light scanning microscope excitation optic capable of focusing the scanned excitation beam on the sample holder such that a sample within the sample holder is raster-scanned in a pre-determined path, and
a light scanning microscope detector defining at least one light scanning detection focal plane and capable of detecting an excitation generated signal contrast from the sample within the sample holder, the light scanning microscope detector being disposed such that the axis of detection of the at least one light scanning detection focal plane is substantially parallel to the excitation beam path as it intersects the sample;
a light sheet microscope unit comprising;
a light sheet microscope excitation optic capable of using the scanned excitation beam to produce a substantially two-dimensional sample excitation region having an effectively uniform excitation intensity which extends in the direction of an excitation axis of the excitation beam path and transversely thereto and intersects with at least a portion of the sample holder, and
a light sheet microscope detector defining at least one light sheet detection focal plane and capable of detecting an excitation generated signal contrast from the sample excitation region, the light sheet microscope detector being disposed such that the axis of detection of the at least one light sheet detection focal plane is substantially orthogonal to the sample excitation region; and
an optical relay disposed downstream of said scanning optic, said optical relay capable of being removably interposed into the excitation beam path, such that said excitation beam may be directed either toward the light scanning microscope excitation optic or the light sheet microscope excitation optic.

### Claim 2 (depends on 1)

The microscope as claimed in claim 1, wherein the optical relay is a periscope having a 4f optical configuration.

### Claim 3 (depends on 1)

The microscope as claimed in claim 1, wherein at least one excitation source is a continuous-wave laser producing an excitation beam in the visible wavelength range, said excitation source being capable of inducing 1-photon-exicted signal contrast for imaging, and wherein said signal contrast is proportional to In, where I is the instantaneous intensity of the excitation beam at the sample excitation region and n is the number of photons involved in the excitation, and where n is 1.

### Claim 4 (depends on 1)

The microscope as claimed in claim 1, wherein the at least one excitation source is capable of producing an excitation beam of high enough intensity to induce sufficient levels of multi-photon excitation to produce signal contrast for imaging, and wherein said signal contrast is proportional to In, where I is the instantaneous intensity of the excitation beam at the sample excitation region and n is the number of photons involved in the multi-photon excitation, and where n is greater than 1.

### Claim 5 (depends on 1)

The microscope as claimed in claim 1, wherein the excitation source is a pulsed near-infrared laser selected from the group consisting of lasers having pulse durations in the range of nanosecond, picoseconds, and femtosecond.

### Claim 6 (depends on 1)

The microscope as claimed in claim 1, wherein the detected signal contrast is selected from the group consisting of 1-photon-excited fluorescence, Rayleigh scattering, Raman-shifted scattering, second harmonic generation, third harmonic generation, sum frequency generation, and stimulated Raman scattering.

### Claim 7 (depends on 1)

The microscope as claimed in claim 1, wherein the at least one excitation source is capable of creating two coaxial and oppositely aligned excitation beams, such that said sample excitation region is formed from the overlap of said two excitation beams.

### Claim 8 (depends on 1)

The microscope as claimed in claim 1, wherein the numerical aperture of at least one of either the light scanning microscope excitation optic or the light sheet microscope excitation optic is adjustable.

### Claim 9 (depends on 8)

The microscope as claimed in claim 8, wherein the adjustable numerical aperture comprises a beam expander with an adjustable expanding ratio.

### Claim 10 (depends on 1)

The microscope as claimed in claim 1, wherein the numerical aperture of at least one of either the light scanning microscope excitation optic or the light sheet microscope excitation optic is anisotropic along at least two axes of said excitation beam.

### Claim 11 (depends on 1)

The microscope as claimed in claim 1, wherein focal volume engineering is applied to the excitation beam to optimize for light sheet imaging.

### Claim 12 (depends on 11)

The microscope as claimed in claim 11, wherein focal volume engineering is implemented using one of the techniques selected from the group consisting of having the numerical aperture of the excitation focusing optics being anisotropic along at least two axes of said excitation beam, and having the excitation beam be a Bessel beam.

### Claim 13 (depends on 11)

The microscope as claimed in claim 11, wherein the focal engineering is implemented by one or more optical elements selected from the group consisting of two orthogonally oriented sequential adjustable slit apertures, a plurality of independently expanding beam expanders, liquid crystal spatial light modulators, digital micromirror device spatial light modultators, and axiconic lens.

### Claim 14 (depends on 1)

The microscope as claimed in claim 1, wherein the sample holder is moveable relative to the sample excitation region along or about at least one axis.

### Claim 15 (depends on 1)

The microscope as claimed in claim 1, wherein the sample excitation region is moveable relative to the sample holder along or about at least one axis.

### Claim 16 (depends on 1)

The microscope as claimed in claim 1, wherein the excitation beam is one of either substantially planar-shaped or linearly-shaped.

### Claim 17 (depends on 1)

The microscope as claimed in claim 1, wherein the light sheet microscope unit incorporates a technique selected from the group consisting of Orthogonal Plane Fluorescence Optical Sectioning (OPFOS), Selective Plane Illumination Microscopy (SPIM), Ultramicroscopy, and Digital Scanned Laser Light Sheet Fluorescence Microscopy (DSLM).

### Claim 18

A method of imaging an object using a dual-mode imaging microscope comprising:
producing an excitation beam, and directing said excitation beam along an excitation beam path;
scanning said excitation beam along a predetermined path;
placing a sample within the optical path of said excitation beam to generate a signal contrast;
directing said excitation beam into an optical relay capable of selecting between one of either a light scanning mode or a light sheet mode;
wherein in the light scanning mode, the method further comprises:
focusing the scanned excitation beam to a point scanning region such that the sample is raster-scanned by the excitation beam to produce a signal contrast in said sample, and
detecting said signal contrast along a detection axis that is substantially parallel to the excitation beam; and
wherein in the light sheet mode, the method further comprises:
focusing said scanned excitation beam to produce a substantially two-dimensional sample excitation region having an effectively uniform excitation intensity which extends in the direction of an excitation axis of the excitation beam path and transversely thereto and intersects with at least a portion of the sample holder, and
detecting said signal contrast along a detection axis that is substantially orthogonal to the sample excitation region.

### Claim 19 (depends on 18)

The method as claimed in claim 18, wherein the optical relay is a periscope having a 4f optical configuration.

### Claim 20 (depends on 18)

The method as claimed in claim 18, wherein at least one excitation beam is produced with a continuous-wave laser producing an excitation beam in the visible wavelength range, said excitation source being capable of inducing 1-photon-exicted signal contrast for imaging, and wherein said signal contrast is proportional to In, where I is the instantaneous intensity of the excitation beam at the sample excitation region and n is the number of photons involved in the excitation, and where n is 1.

### Claim 21 (depends on 18)

The method as claimed in claim 18, wherein the at least one excitation beam is of high enough intensity to induce sufficient levels of multi-photon excitation to produce signal contrast for imaging, and wherein said signal contrast is proportional to In, where I is the instantaneous intensity of the excitation beam at the sample excitation region and n is the number of photons involved in the multi-photon excitation, and where n is greater than 1.

### Claim 22 (depends on 18)

The method as claimed in claim 18, wherein the excitation beam is produced via a pulsed near-infrared laser selected from the group consisting of lasers having pulse durations in the range of nanosecond, picoseconds, and femtosecond.

### Claim 23 (depends on 18)

The method as claimed in claim 18, wherein detecting the signal contrast includes using a detection technique selected from the group consisting of fluorescence, second harmonic generation, third harmonic generation, sum frequency generation, and stimulated Raman scattering.

### Claim 24 (depends on 18)

The method as claimed in claim 18, further comprising forming at least two coaxial and oppositely aligned excitation beams, such that said sample excitation region is formed from the overlap of said two excitation beams.

### Claim 25 (depends on 18)

The method as claimed in claim 18, wherein the focusing of the excitation beam further includes adjusting the numerical aperture of a focusing optic.

### Claim 26 (depends on 18)

The method as claimed in claim 18, wherein the focusing of the excitation beam further includes anisotropically adjusting the numerical aperture of a focusing optic such that the excitation beam is anisotropic along at least two axes.

### Claim 27 (depends on 18)

The method as claimed in claim 18, wherein the imaging is performed in one of either a 3D or 4D mode.