Background free imaging in stimulated emission fluorescence microscopy

Abstract

Recently, the pump-probe technique has found highly valued developments and applications in optical microscopy to study both labeled (fluorescence) and non-labeled (vibrational) for observation of cells and tissues. In this work, we use the mechanism of stimulated emission (SE) with dual frequency modulation to remove the large background signal that is originated from spontaneous emission. We have applied a high performance digital signal processing based lock-in detection for stimulated emission fluorescence microscopy. Critically, the pump beam is modulated at f1 and the probe beam is at a different frequency, f2, with the demodulation carried out at the sum of the two frequencies, (f1 + f2). In this way, the DC background is effectively removed. Note that the background is mainly attributed to the spontaneous emission of the pump beam, with modulated frequency f1.

Advances in nonlinear optical microscopy techniques for in vivo and in vitro neuroimaging

“…SE was first used by Hell and Wichmann ( 1994 ) to achieve beyond diffraction-limited resolution in light microscopes. In recent years, several SE based optical imaging modalities have emerged for the detection of the so-called undetectable chromophores (Min et al 2009 ) and fluorescently labeled samples (Das et al 2019a ; Das et al 2019b ). However, fluorescence labeling can be difficult in some small intracellular molecules such as signaling peptides, neurotransmitters, and metabolites as the size of the fluorescent molecule is much larger than that of the molecule of interest.…”

Dual-modulation difference stimulated emission depletion microscopy to suppress the background signal

“…18 Also, dual modulation with lock-in detection has long-term compatibility to pump-probe microscopy. 19,20 To date, a phasor-domain technique, called separation of photons by lifetime tuning (SPLIT), explores analysis of lifetimes to separate fluorescence in the center of the PSF, fluorescence in the periphery of the PSF, and STED-induced background. [21][22][23][24][25][26] Sarmento et al 22 performed dual temporal modulation of excitation and depletion beams and implemented an analysis in the phasor plot at a single frequency.…”

High temporal resolution and polarization resolved fluorescence lifetime measurements through stimulated emission

“…The well-known non-linear optical transient absorption and gain processes include two-photon absorption (TPA) [4][5][6], excited-state absorption (ESA) [5], ground-state depletion (GSD) [7], and stimulation emission (SE) [8]. Among these nonlinear optical processes, SE exhibits the novelties of subdiffraction imaging [9], detection of 'dark' (nonfluorescent) fluorophores [10], fluorescence imaging at an extended working distance [11][12][13], and polarization-resolved (PR) lifetime measurements of fluorophore [14]. SE signal is usually detected via lockin technique, in which the incident or pump beam is modulated and the non-linear signal of interest is extracted by demodulating the probe beam.…”