SG182865A1

SG182865A1 - Flexible spatial-temporal phase modulator for focal modulation microscopy

Info

Publication number: SG182865A1
Application number: SG2011002334A
Authority: SG
Inventors: Nanguang Chen
Original assignee: Univ Singapore
Priority date: 2011-01-13
Filing date: 2011-01-13
Publication date: 2012-08-30
Also published as: WO2012096629A1

Abstract

Flexible spatial-temporal phase modulator for focal modulation microscopy AbstractA spatial-temporal phase modulator (SPM) for modulating the phase of a collimated light beam spatially and temporally. The collimated beam can be focused by a lens and the intensity within the focal volume can be temporally modulated.Figure 1.

Description

Titie

Flexible spatial-temporal phase modulator for focal modulation microscopy.

Field of invention

This invention is related to laser scanning optical microscopy, in particular focal modulation microscopy.

Backarcund of invention

Confocal microscopy (CM) is the standard tool widely used for the imaging of thick biological slices. However CM suffers from poor resolution due to noise signals from back-scattered light. To overcome the limitation of CM, Focal Modulation Microscopy (FMM) was developed as a novel imaging method for improved imaging depth. Focal

Modulation Microscopy (FMM) combines focal modulation with confocal detection to reject background signal from scattering media. It has demonstrated a much improved imaging depth than confocal microscopy (CM) [1-3]. The difference between the FMM and the CM is a spatial-temporal phase modulator (SPM) introduced into the illumination beam path, which results in an intensity modulation in the focal volume where the modulated beam is focused by an objective. The modulated component in the detected signal is related to the ballistic excitation light only and can be differentiated easily from the background by the use of lock-in detection.

To ensure proper lock-in detection, the pixel dwell time should be an integer multiple of the modulation period. In modern commercial laser scanning microscopes, the pixel dwell time is usually less than 10 microseconds, which implies that the modulation frequency should be greater than 100 kHz. Two simple spatial-temporal phase modulators have been designed and implemented for the prototype FMM system [1-2].

However, their modulation frequencies are limited by a few kHz and the corresponding image acquisition time is at least tens of seconds. A high-speed design is proposed recently, which is based on acousto-optical modulators (AOM). The schematic of the

FMM with such a modulator is shown in Fig. 1. The coherent laser beam is first split by a beam splitter into two beams which then passes through two AOMs with slightly different resonance frequencies (ie. f1 and f2) where they undergo zeroth- and first-order diffractions. The first-order diffracted beams are Doppler shifted by f1 or f2. They are reflected with slight lateral displacement using retroreflectors to return to the same

AOMs and then are recombined at the beam splitter. it is important to note that the two beams at the moment are Doppler shifted at twice the resonance frequencies of the

AOMs respectively as they passed through the AOMs twice. Part of the combined beam is directed fowards a fiber-optic photodetector, which generates a reference signal at the optical beating frequency of 2(f1 - £2). The remaining part of the modulated laser beam is directed to the scanning unit of a conventional CM to excite the biological sample stained with fluorescence probes. The fluorescence emissions are then detected by the

PMT behind a confocal pinhole. The PMT output is preamplified before feeding to an 1/Q demodulator, where the osciliatory component at the beating frequency is picked up by mixing with the reference signal. The demodulated signal is then used to form the FMM images. The typical frequency of AOMs is tens of MHz. A 10 MHz beating frequency can be readily achieved, which corresponds to a minimum pixel dwell time of C.1 microsecond.

While the spatial-temporal phase modulator shown in Fig. 1 is fast enough for real-time image acquisition, its aperture (Fig. 2) is far from optimized. Only part of the objective aperture is occupied by the two ACM modulated beams. This leads to deteriorated spatial resolution, and more significantly, a small modulation depth. Modulation depth is a very important parameter in FMM, which is defined as the ratio of the amplitude of the ac component to the magnitude of the dc component in the detected fluorescence signal:

M = Lf, in practical situations, the excitation power needs to be maintained at the lowest possible level to reduce photobleaching and phototoxicity. In addition, [, contributes to shot noise which cannot be completely eliminated by filtering. Consequently, it is desirable to maximize the modulation depth M. The measured modulated depth for the aperture shown in Fig. 2 is only about 0.25. The inventor has conducted theoretical investigations to evaluate the modulation depth for various aperture configurations, as shown in Fig. 3. It is found that the six-zone annual aperture provides the best performance (M~0.82) while the double-D aperture is the worst (M~0.35). While multi- zone apertures are preferred for a large M and enhanced FMM signal, such configurations are difficult to implement using the existing approach.

References:

[1] N. Chen, C-H. Wong, C. J. R. Sheppard, “Focal modulation microscopy,” Optics

Express 16 (23), 18764-18769 (2008).

[2] C. H. Wong, S. P. Chong, C. J. R. Sheppard, and N. Chen, “Simple spatial phase modulator for focal modulation microscopy,” Applied Optics 48 (17), 3238-3243 (2009).

[3] S. P. Chong, C. H. Wong, C. J. R. Sheppard, and N. Chen, “Focal Modulation

Microscopy: A Theoretical Study,” Optics Letters 35 (11), 1804-1806 (2010).

Summary of invention

Focal modulation microscopy (FMM) is a novel imaging method for improved imaging depth. A spatial-temporal phase modulator is used in the focal modulation microscope to generate an intensity modulation at the focal point, which is critical for improving the signal to background ratio. The desired characteristics of the spatial-temporal phase modulator include high-speed, optimal aperture, and compatibility with multiple wavelengths. Such properties, however, are not readily available with commercial products and existing designs. This invention provides an integrated solution that overcomes the limitations of existing designs. The novel spatial-temporal phase modulator features an efficient combination of high-speed temporal moduiators with polarization optics. Arbitrary aperture phase distribution can be implemented with time- dependent modulation fast enough for real-time imaging acquisition.

Brief description of drawings

Fig. 1: A FMM with acousto-optical modulators (AOM). Laser beam is split by a beam splitter (BS) in which light beam in each arm undergoing frequency shifting by two AOMs with different resonance frequencies. Phase-shifted beams are reflected back to the BS by retroreflectors (R1 and R2). They are aligned in parallel non-overlapping manner when being split second times at BS in which one combined beam will be used to generate a reference signal while another combined beam is directed to the scanning unit of a CM to excite the samples through the objective lens (OBJ). PD is a photodetector and M is a mirror.

Fig. 2: Effective aperture of two AOM modulated beams.

Fig. 3: Configurations of SPM apertures. Rays passing a gray zone and a white zone have different phase delays.

Fig. 4: Single EOM based spatial-temporal phase modulator. HWP: Half wave plate; PA:

Polarization analyzer, SP: Spatial polarizer.

Fig. 5: Configuration of a four-zone spatial polarizer.

Detailed description of invention

A novel design of high-speed SPM is described in the following. in such a modulator, there are two orthogonally polarized beams which are modulated differently with high- speed temporal phase modulators. These two beams are parallel to each and they are spatially overlapping before entering the aperture forming optics. The aperture forming optics includes a spatial polarizer that allows only one polarization state to pass through a specific area. The excitation beam after the aperture forming optics is spatial-temporal modulated with desired properties. In the basic embodiment, a single eletro-optical modulator (EOM) is combined with polarization optical components (Fig. 4). The laser output is linearly polarized and a half wave plate (HWP) is used to rotate the polarization of the E-field fo form a 45 degree angle with the Y-axis. The two orthogonally polarized components, Ex and Ey, carry identical power. The EOM is a polarization dependent device. It provides a variable phase shift on Ey and but no phase shift on Ex. A RF signal (~MHz) is fed to the EOM to introduce a periodic phase delay (between 0 to 1) between

Ex and Ey. The aperture-forming optics consists of a spatial polarizer (SP) and polarization analyzer (PA). The spatial polarizer selectively blocks Ex or Ev so that the modulated and non-modulated beams are spatially separated in the output. A four-zone annular spatial polarizer is illustrated in Fig. 5. The gray zones allow veriically polarized light (Ev) to pass while the white zones pass horizontally polarized light (Ex). The PA axis is at 45 degrees with the polarization directions of both modulated and non- modulated beams so that they can interfere with each after the PA.

The spatial-temporal phase modulator, consisting of the HWP, EOM, SP, and PA, has many advantages. First of all, The EOM modulation frequency can easily reach a few

MHz range. Secondly, the aperture is defined by the spatial polarizer, which is easy to configure and fabricate. Thirdly, the modulator can be shared by multiple excitation wavelengths simultaneously. Fourthly, such a design has a very high ievel flexibility. The aperture forming optics is very compact and can be easily inserted into the scan head of a standard confocal microscope, while the EOM can be integrated info the [aser system.

The output of the EOM can be linked to the aperture forming optics via a polarization maintaining fiber. Last but not least, the aperture forming optics can be miniaturized and integrated into an endoscopic imaging catheter.

What problems does the invention solve and advantages over existing methods, devices or materials?

The spatial-temporal phase modulator is a critical part of the focal modulation microscope. Its design and properties directly affect the imaging performance. A desirable SPM should be able to generate a modulated excitation beam at a frequency greater than 100 kHz, and the aperture should lead to a large modulation depth.

Commercially available phase modulators are either too slow or not capable of spatial modulation. Compared with previously proposed solutions, this invention has the following advantages: 1) High modulation frequency that is adequate for real-time imaging. 2) Optimal apertures can be easily implemented. 3) Great ease to upgrade existing confocal microscopes. 4) Compatible with multiple excitation wavelengths. 5) Ready to be miniaturized.

What are the possible specific industrial applications?

The invention can be applied to the newly invented focal modulation microscopy.

Does the invention possess any disadvantages or limitations? Can they be overcome?

What are the competing ways to solve the same problems?

The invention has no obvious disadvantage or limitation. Other approaches do not provide comparable performances.

Modifications of the preferred embodiments:

Alternative embodiment 1:

The aperture forming optics can be placed afier the scanner and before the objective.

Alternative embodiment 2: in the aperture forming optics, the spatial polarizer can be replaced by a spatial retarder. Part of incident beam has its polarization rotated by 90 degrees while the rest of beam remains unchanged. The spatial retarder may be followed by a polarizer.

Alternative embodiment 3:

The two orthogonally polarized excitation beams can be temporally modulated by AOMs.

Claims

Claims: A summary is of the technology disclosure is provided below:

1. A spatial-temporal phase modulator (SPM) for modulating the phase of a collimated fight beam spatially and temporally. The collimated beam can be focused by a lens and the intensity within the focal volume can be temporally modulated.

2. A method of claim 1 used in the focal modulation microscope (FMM) to generate an intensity modulation at the focal point for improving the signal to background ratio.

3. A method of claim 2 having a high modulation depth value, defined as the ratio of the amplitude of the ac component to the magnitude of the dc compenent in the detected fluorescence signal.

4. A high-speed design of claim 2 in excess of several MHz range enabling imaging capture for display in real-time.

5. A method of claim 2 that is compatible with multiple excitation wavelengths.

6. A system of spatial-temporal phase modulator of claim 1 featuring a combination of high-speed temporal modulators with polarization opiics.

7. A system of spatial-temporal phase modulator of claim 1 featuring arbitrary segmentation of the aperture and fast time-dependent phase modulation within individual segments.

8. A system of spatial-temporal phase modulator of claim 1 featuring two orthogonally polarized beams which are modulated differently with high-speed temporal phase modulators.

9. These two beams of claim 8 are parallel to each other and they are spatially overlapping before entering the aperture forming optics.

10. The aperture forming optics of claim 9 includes a spatial polarizer that allows only one of the two polarization states to pass through a specific aperture segment, and a polarization analyzer.

11. The excitation beam of claim 10 after the aperture forming optics is spatial-femporal modulated with desired properties.

12. In an embodiment of claim 10 a single eletro-optical modulator (EOM) is combined with polarization optical components.

13. The input light polarization of claim 12 is arranged to form a 45 degree angle with the fast axis of EOM, which a polarization dependent device.

14, An RF signal (in the range of a few MHz) is fed fo the EOM of claim 12 to introduce a periodic phase delay (between 0 to 1m) between the ordinary and extraordinary waves.

15. The aperture-forming optics of claim 12 consists of a spatial polarizer (SP) and polarization analyzer (PA). The spatial polarizer selectively blocks the ordinary wave or the extraordinary wave so that the modulated and non-moduiated beams are spatially separated in the output.

16. A polarization analyzer (PA) of claim 10 having its axis at 45 degrees with the ordinary and extraordinary waves so that they can interfere with each other after the PA.