Research Progress and Prospects of Low‑Power Stimulated Emission Depletion Microscopy

Haoxian Zhou; Luwei Wang; Renlong Zhang; Fangrui Lin; Liwei Liu; Junle Qu

doi:10.3788/CJL240959

Chinese Journal of Lasers, Volume. 51, Issue 21, 2107101(2024)

Research Progress and Prospects of Low‑Power Stimulated Emission Depletion Microscopy

Haoxian Zhou, Luwei Wang, Renlong Zhang, Fangrui Lin, Liwei Liu, and Junle Qu^*

Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education/Guangdong Province, College of Physics and Optoelectronic Engineering, Shenzhen University, Shenzhen 518060, Guangdong , China

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Figures & Tables(6)

Fig. 1. Schematic diagrams of STED. (a) Imaging principle diagram; (b) simplified Jablonski energy level diagram for a typical STED process; (c) Atto 647N dye spectra

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Fig. 2. Schematic diagrams of low-power STED imaging methods based on single molecular localization. (a) MINFLUX^[83];

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Fig. 3. Schematic diagrams of low-power STED imaging methods based on image signal processing. (a) DE-STED^[92]; (b) mSTED^[93]; (c) FM-STED^[94]

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Fig. 4. Schematic diagrams of low-power STED imaging methods based on time-resolved detection. (a) Time-gating^[101]; (b) SPLIT^[97]; (c) phasor plot analysis^[98]; (d) ratiometric photon reassignment^[99]

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Table 1. Parameters and imaging indexes of STED probes (λ_ex: excitation wavelength; λ_em: emission wavelength; I_sat: saturation intensity; P_sat: STED power; τ: fluorescence lifetime)

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Table 1. Parameters and imaging indexes of STED probes (λ_ex: excitation wavelength; λ_em: emission wavelength; I_sat: saturation intensity; P_sat: STED power; τ: fluorescence lifetime)

Type	Original name	λ_ex & λ_em / nm	I_sat（P_sat）	τ	Photostability	Resolution / nm
Organic dyes	Abberior STAR 488^［34］	503 & 524		3.9 ns		110
	Atto 532^［76］	532 & 552		3.8 ns		~20
	Atto 647N^［16］	644 & 669	9 mW	3.5 ns		52
	MitoPB-Yellow^［41］	480 & 570	1.3 MW/cm²	7.5 ns	Over 390 s	<60
	MitoESq-635^［42］	635 & 670	4.37 MW/cm²	1.7 ns	Over 50 min	35.2
	520R^［43］	521 & 546		4.0 ns		40
	Complex1^+［45］	488 & 660		180 ns	Over 1000 s	35（XY） 120‒150（Z）
AIE dots	TPA-T-CyP^［56］	480 & 684	~50 mW		Over 17.5 s	74
	Red-AIE-OXE^［57］	405 & 640		4.5 ns		95
	TTF@SiO₂^［58］	510 & 660	40 mW	1.29 ns	The fluorescence intensity decreased by 30% after one and a half hours of imaging	30.7
PDs	PDFDP^［60］	403 & 633	~0.4 mW		The fluorescence intensity decreased by 30% after two hours of imaging	71
PDs	CNPPV^［77］	455 & 604	~0.5 mW		The fluorescence intensity decreased by 44% after two hours of imaging	78
Other types of organic nano-particles	CSONPs^［61］	500 & 650	0.0085 MW/cm²		Over 600 s	60
	Organosilica nanohybrids^［75］	470 & 520	0.188 MW/cm²	4.37 ns	The fluorescence intensity decreased by 50% after 100 scans	43.6
	DBTBT-4C8^［62］	496 & 655	11.9 mW	3.85 ns	Over 25 min	100
QDs	Qdots705^［78］	445 & 705	~50 mW	8 ns	The fluorescence intensity decreased by 50% after 1300 scans	~50
QDs	CsPbBr₃^［64］	488 & 520	0.126 MW/cm²	15.5 ns	Over 200 min	20.6
UCNPs	8%Tm³⁺+ 20% Yb^3+［65］	980 & 455	0.19 MW/cm²	Tens of μs to ms	Over 200 min	28
UCNPs	10% Tm³⁺+ 18% Yb^3+［66］	975 & 455	0.849 MW/cm²	Tens of μs to ms	Over 3000 scans	66
CDs	FNCDs^［70］	466 & 513	0.226 MW/cm²	4.89 ns	The fluorescence intensity decreased by 12% after 1000 scans	22.1
CDs	CPDs-3^［72］	465 & 515	0.23 MW/cm²	4009 ps	The fluorescence intensity decreased by 30% after 200 scans	92
FNDs	NV center^［79］	550 & 685		13 ns		4.1
FNDs	NVN center^［80］	480 & 530	2.5 MW/cm²	27 ns	Over 100 scans	90
LPR hybrids	AuNRs^［73］	610 & 640	~15 MW/cm²	3.5 ns±0.2 ns		Up to 50% increase
LPR hybrids	Gold nanospheres^［81］	500 & 525	4.6‒5.8 MW/cm²	0.95 ns	The fluorescence intensity decreased by 50% after 20 scans	75

Table 2. Imaging performance comparison of STED and its derivative techniques

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Table 2. Imaging performance comparison of STED and its derivative techniques

Method	Type of STED	Resolution	Advantage	Disadvantage
Conventional STED	pSTED^{［98，109］}	40 nm fluorescent beads： 52 nm/24 mW； endoplasmic reticulum （PTK2）： 52 nm/36 mW	Higher peak power compared to CW-STED for the same resolution at lower depletion power	High cost and complexity； need pulse synchronization
Conventional STED	CW-STED^［16］	20 nm fluorescent beads： 34 nm/812 mW； neurofilaments： 52 nm/432 mW	Low cost and complexity； wide wavelength coverage	The average power is 4‒5 times higher compared to pSTED， increasing the probability of photobleaching and re-excitation
MINFLUX	pSTED^［83］	~1 nm precision； ~5 nm resolution	The localization precision and imaging resolution are greatly improved	Complex controls and algorithms are required
LocSTED	CW-STED^［84］	Single molecules of Alexa 555： ~15 nm	Offer a more relaxed requirement for STED beam to achieve high-resolution	Require specialized imaging buffers for the reversible blinking of fluorophores and careful control to achieve optimal results
MINSTED	pSTED^［87］	Ångström range precision； ~4 nm resolution	Higher resolution and more efficient particle discovery compared to MINFLUX	The high depletion power for precise localization causes anti-Stokes background noise
On-line time-gated STED	CW-STED^［102］	40 nm fluorescent beads： 35 nm/250 mW； microtubule （PtK2）： 89 nm/300 mW	Simple operation， effective resolution improvement （better resolution improvement for CW-STED）	Require precise time synchronization； reduce the SNR （signal to noise ratio） of image
On-line time-gated STED	pSTED^［102］	Microtubule （PtK2）： 89 nm/45 mW
Off-line time-gated STED	CW-STED^［100］	Microtubule （astrocytes）： 70 nm/85 mW	Reduce system costs and flexibly set time gates	Reduce the SNR of image
SPLIT	CW-STED^［97］	Microtubule （HeLa）： $~$ 100 nm/40 mW；	Improve resolution without affecting the SNR	The separation criteria required to improve resolution is not quantified； the accuracy of photon separation is affected by discrete noise
SPLIT	pSTED^［107］	60 nm fluorescent beads： 69 nm±4 nm/25 mW	Improve resolution without affecting the SNR
PPA	pSTED^［98］	100 nm fluorescent beads： 118 nm/10 mW； HeLa： 86 nm/20 mW	The resolution improvement effect is better than the time-gating	TCSPC is required for PPA， complicating the system
RPR-STED	pSTED^［99］	40 nm fluorescent beads： 96 nm/60 mW； microtubule （HeLa）： 108 nm/50 mW	Improve the resolution without affecting the SNR； TCSPC is not required for PPA， simplifying the system	The resolution improvement effect is not obvious compared to time-gating
DE-STED	pSTED^［92］	23 nm fluorescent beads： 30 nm±8 nm/3.6 mW； microtubule （BSC-1）： 82 nm/1.4 mW	The depletion power is greatly reduced	An image mismatch may arise owing to sample drift
mSTED	pSTED^［93］	23 nm fluorescent beads： 41.2 nm±8 nm/1.95 mW； microtubule （HeLa）： 90 nm/1.8 mW	Solve the pixel mismatch problem	Susceptible to aberration
FM-STED	pSTED^［94］	23 nm fluorescent beads： 140 nm / 10 mW； microtubule （BSC-1）： 108 nm / 10 mW	Effectively improve resolution and remove background signals	The fluorescence intensity decreased after treatment

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Haoxian Zhou, Luwei Wang, Renlong Zhang, Fangrui Lin, Liwei Liu, Junle Qu. Research Progress and Prospects of Low‑Power Stimulated Emission Depletion Microscopy[J]. Chinese Journal of Lasers, 2024, 51(21): 2107101

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Paper Information

Category: Biomedical Optical Imaging

Received: Jun. 13, 2024

Accepted: Jul. 4, 2024

Published Online: Oct. 31, 2024

The Author Email: Qu Junle (jlqu@szu.edu.cn)

DOI:10.3788/CJL240959

CSTR:32183.14.CJL240959

Topics

laser devices and laser physics

Lasers and Laser Optics

Laser physics

laser manufacturing

Instrumentation, Measurement and Metrology

Table 1. Parameters and imaging indexes of STED probes (λex: excitation wavelength; λem: emission wavelength; Isat: saturation intensity; Psat: STED power; τ: fluorescence lifetime)

Table 1. Parameters and imaging indexes of STED probes (λex: excitation wavelength; λem: emission wavelength; Isat: saturation intensity; Psat: STED power; τ: fluorescence lifetime)

Table 2. Imaging performance comparison of STED and its derivative techniques

Table 2. Imaging performance comparison of STED and its derivative techniques

Table 1. Parameters and imaging indexes of STED probes (λ_ex: excitation wavelength; λ_em: emission wavelength; I_sat: saturation intensity; P_sat: STED power; τ: fluorescence lifetime)

Table 1. Parameters and imaging indexes of STED probes (λ_ex: excitation wavelength; λ_em: emission wavelength; I_sat: saturation intensity; P_sat: STED power; τ: fluorescence lifetime)