The combination of genetically modified fluorescent proteins and exogen
ous fluorescent dyes is a widely used imaging technique in biological and
materials research due to its precise labeling capability for targeting speci
fic neurons. Fluorescence provides high sensitivity and background-free d
etection. However, many molecules do not naturally emit fluorescence, re
quiring genetic modification or staining for imaging. Fluorescent labeling
can perturb samples, especially small molecules like DNA, RNA, and prote
ins, hindering the understanding of their original functional states. Additi
onally, fluorescence suffers from photobleaching issues under prolonged
or intense laser illumination, limiting long-term observations. To address t
hese limitations, we propose using coherent Raman scattering (CRS) micr
oscopy for label-free imaging of neuronal responses and molecular dyna
mics. CRS microscopy combines high sensitivity, spatial resolution, and m
olecular identification capabilities by observing unique Raman signal characteristics. CRS signals are generated by the precise overlap of two short-
pulsed laser beams with distinct wavelengths. Unlike traditional systems,

we utilize a single coherent supercontinuum light source with a tunable w
avelength filter, providing dual-color pulses for Raman imaging. This light
source enhances and improves various aspects of CRS performance: (1)Th
e supercontinuum light source covers the entire Raman-active region (0-4
000 cm-1), enabling the observation of different molecules below 1000 c
m-1 within biological organisms. (2)The two coherent wavelengths can be
independently adjusted, allowing the induction of electronic preresonanc
e (EPR) modes to enhance the Raman signal and improve sensitivity. (3) A
ble to simultaneously observe multiple Raman shifts, enhancing the specif
icity. This technique has been successfully applied to biological imaging of
Drosophila brains, and EPR Raman signal enhancement by a factor of >10
0 in ch