Compact simultaneous label -free autofluorescence multi -harmonic SLAM microscopy for u ser- friendly photodamage -monitored imaging

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Compact simultaneous label-free autofluorescence

multi-harmonic (SLAM) microscopy for user-

friendly photodamage-monitored imaging

GENG WANG,1 STEPHEN A. BOPPART, 1,2,3,4,5,6 AND HAOHUA TU 1,2,*

1 Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana,

Illinois 61801, USA

2 Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign, Urbana, IL

61801, USA

3 Department of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

4 Cancer Center at Illinois, Urbana, IL 61801, USA

5 Carle Illinois College of Medicine, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

6 Center for Label-free Imaging and Multi-scale Biophotonics (CLIMB), Urbana, IL 61801, USA

* htu@illinois.edu

Keywords: Multiphoton microscopy, multimodal imaging, photodamage, tunable ultrashort pulse, high-

speed imaging.

Abstract: Label-free nonlinear optical microscopy has become a powerful tool for biomedical research.

However, the possible photodamage risk hinder further clinical applications. To reduce these adverse

effects, we constructed a new platform of simultaneous label-free autofluorescence multiharmonic

(SLAM) microscopy, featuring 5-channel multimodal imaging, inline photodamage monitoring, and

pulse repetition-rate tuning. By the use of a birefringent photonic crystal fiber for spectral broadening

(rather than supercontinuum generation) and a prism compressor for pulse pre-chirping, this system

allows users to independently adjust pulse width, repetition rate, and energy, which is useful for

optimizing imaging condition towards no/minimal photodamage. Also, it demonstrates label-free

multichannel imaging at one excitation pulse per image pixel and thus paves the way for improving the

imaging speed by a faster optical scanner.

1. Introduction

Intravital imaging technology with high speed, sufficient spatial resolution, and long-term photodamage-

free capability is critical to the study of biological processes. It permits direct and longitudinal tracking

of diverse intercellular behaviors in their native environment instead of inferring possible processes

based on static images [1-4]. As one type of intravital imaging, label-free nonlinear optical microscopy

[5-8] has become a powerful tool for biomedical research due to its advantages of low invasiveness, deep

imaging penetration, high resolution, etc., especially in neuroscience, oncology and immunology [9-12].

However, the relatively slow imaging speed and the accompanying photodamage risk bring limitations

to further preclinical and clinical studies. The improvement of imaging speed and the suppression of

photodamage can be achieved by different methods, such as improving the excitation efficiency [7,13],

using a polygonal mirror and resonant scanner [14], splitting one pulse to sub-pulses with equal energy

[15], applying an adaptive light source to illuminate only the area of interest (ROI) [16], or improving

the signal-to-noise ratio(SNR) of a single frame through a deep learning algorithm [17,18]. One

fundamental factor that limits the imaging speed is the low excitation efficiency of a single pulse. For a

typical 80 MHz imaging system [19], the power of each laser pulse has to be kept low in order to avoid

heating-related photodamage. Therefore, in order to obtain an image with acceptable SNR, each pixel

needs to contain tens to hundreds of pulses, which greatly limit the imaging speed.

In 2018, by a combination of reduced repetition rate of the laser source (10 MHz) and shortened

pulse width (<60 fs) at 1110-nm central wavelength, You et al. obtained a relatively high peak power of

optical pulses and demonstrated simultaneous label-free autofluorescence multi-harmonic (SLAM)

microscopy [7], which can simultaneously acquire the signals of two- and three-photon excited

autofluorescence (2PAF/3PAF) of FAD/NAD(P)H, respectively, and second/third harmonic generation

(SHG/THG), by using the supercontinuum generation technology based on a photonic crystal fiber (PCF).

However, several limitations remained: 1) The high peak power supercontinuum generation leads to a

short life of the PCF (~200 hr), which needs to be replaced regularly and thus complicates the operation

of the system; 2) The limited average power for a given excitation band (~60 nm) does not allow users

to adjust pulse repetition rate in a wide range to meet different imaging requirements; 3) Although the

peak power of excitation pulse is greatly improved, the photon-counting photomultiplier (PMT) detection

requires a large number (>10, typically) of excitation pulses for each pixel (a long pixel dwell time) for

sufficient signal (counts), which limits imaging speed; 4) The components used in the system are large,

resulting in a rather bulky and complex system.

Here, we demonstrate a new and compact SLAM platform and system. The central wavelength of

the excitation window is shifted to ~1030 nm, which is much accessible commercially. By using a PCF

and a prism compressor, we obtain excitation pulses from near-transform-limited 60 fs (FWHM) to

uncompressed 300 fs with a broad bandwidth (~80 nm (990-1070 nm), bottom-to-bottom), and sufficient

pulse energy (or average power) under a wide and tunable repetition rate (800 kHz - 20 MHz). More

importantly, these three pulse parameters (width, repetition rate, energy) can be adjusted independently

without interference. This allows users to find the optimal imaging conditions for different applications

to maximize the signal-to-photodamage ratio. With only PCF-assisted spectral broadening free of

supercontinuum generation, the laser source is stable indefinitely (> 1 year) without the need to replace

the PCF. Finally, the higher peak power permits single pulse per pixel label-free imaging via analog PMT

detection, and can greatly increase the imaging speed.

2. Materials and Method

2.1 High excitation efficiency with low duty cycle

For label-free nonlinear imaging, it is critical to achieve high excitation efficiency without photodamage.

According to the nonlinear optical signal generation formula (Eq.1), when the average power P remains

unchanged, the signal generation intensity S can be significantly increased by reducing the duty cycle,

consisting of laser repetition rate f and pulse width τ, especially for high order nonlinear processes:

() ()

Sf ff













(1)

where n denotes the order of nonlinear process (2 for 2PAF/SHG, 3 for 3PAF/THG, 4 for 4PAF). For

two/three/four-photon imaging, when the duty cycle is reduced by a factor of 4, the signal will increase

exponentially and increase by a factor of 4/ 42/ 43, respectively. Therefore, by reducing the duty cycle,

equal or higher signal can be achieved using lower average power (lower risk of thermal damage). More

importantly, it can enable the excitation of higher-order nonlinear processes.

2.2 Excitation window

For this new platform of SLAM microscopy, the central wavelength was shifted to 1030 nm. Compared

with the previous 1110 nm center wavelength, the 1030 nm center wavelength has several advantages: 1)

Relatively short excitation wavelength can provide higher multiphoton excitation efficiency for FAD,

NAD(P)H and tryptophan; 2) The excitation center wavelength is the same as the source laser emission

center wavelength for PCF spectra broadening, so there is no need to use high peak power to generate

supercontinuum, and only need to use low peak power to broaden the spectrum in the PCF to achieve a

near-transform-limited pulse, which can avoid the problem of PCF damage and replacement; 3) Lower

input peak power for PCF allows the same spectral broadening over a wide range of repetition rates (with

a wide range of average power) to meet the needs of different applications,.

2.3 Fiber source

The laser source for PCF spectral broadening is a compact ultrafast laser (Satsuma, Amplitude Laser)

with 1030±5 nm central wavelength (inset a of Fig. 1), 10 W maximum average output power, < 350 fs

pulse width, and tunable repetition rate from single shot to 40 MHz. In order to obtain near-transform

limited pulses to improve the multiphoton signal generation efficiency, laser pulses were first sent into a

25-μm-core PCF (LMA-25, Thorlabs) to achieve a coherent spectral broadening of around 80 nm (1030

± 40 nm, inset a of Fig. 1) with ~80% coupling efficiency, and then the collimated beam was sent to a

prism pulse compressor (BOA-1050, Swamp Optics) for dispersion compensation to obtain 60 fs near-

transform limited pulses, which can be achieved by adjusting the group delay dispersion (GDD) via the

compressor, as shown in the inset b of Fig. 1. In practical application, we should pre-chirp the pulse to

compensate for the dispersion caused by the optical components after the compressor. Compared with

the previous SLAM system [7], the new platform does not need to select an excitation window at 1110 nm

in the supercontinuum, so this eliminates the need of the expensive pulse shaper.

2.4 System setup

We designed a new SLAM platform and system (Fig.1). The 1030 ± 40 nm pulses from the prism

compressor were raster scanned by a 4 mm Galvo-Galvo Scanner (LSKGG4, Thorlabs) and focused by

an inverted high UV-transmission objective (UAPON 40XW340, N.A. = 1.15, Olympus) with an ample

adjustable average power on the sample after the loss along the excitation beam path. One telecentric

scan lens (SL50-2P2, f=50 mm, Thorlabs) and one tube lens (TTL200MP, f=200, Thorlabs) expanded

the beam size to fill the back focal plane pupil of the objective (Ø10.35mm). Since high power is not

required to generate supercontinuum, this system with its PCF can operate over a wide range of repetition

rates (800 KHz~20 MHz) with similar spectra broadening, as shown in the inset a of Fig. 1 (1/2.5/10

MHz). For example, when the repetition rate is 2.5 MHz, the excitation power before PCF is only 180

mW, but the maximum average power on the sample can exceed 50 mW, which gives users a high power

dynamic range for different applications. Based on the objective lens we use, the typical maximum field

of view (FOV) is around 400 μm× 400 μm, the actual FOV depends on the scanning angle of the

galvanometer-driven mirror.

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Compactsimultaneouslabel-freeautofluorescencemulti-harmonic(SLAM)microscopyforuser-friendlyphotodamage-monitoredimagingGENGWANG,1STEPHENA.BOPPART,1,2,3,4,5,6ANDHAOHUATU1,2,*1BeckmanInstituteforAdvancedScienceandTechnology,UniversityofIllinoisatUrbana-Champaign,Urbana,Illinois61801,USA2DepartmentofEl...

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Compact simultaneous label -free autofluorescence multi -harmonic SLAM microscopy for u ser- friendly photodamage -monitored imaging

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