Imaging in turbid media: a transmission detector gives 2-3 order of magnitude enhanced sensitivity compared to epi-detection schemes

Dvornikov, Alexander; Gratton, Enrico

doi:10.1364/boe.7.003747

Cited by 9 publications

(9 citation statements)

References 26 publications

(24 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The autofluorescence of liver sections (5 µm thick) was imaged using the homebuilt DIVER (Deep Imaging via Enhanced Recovery) microscope [24][25][26]. The freshly frozen tissue samples were originally embedded in OCT.…”

Section: Data Acquisition: Diver and Phasor Approach To Flimmentioning

confidence: 99%

“…The microscope used for our measurements is based on a forward direction detector where the data is acquired in the direction of excitation and it is exquisitely sensitive for harmonic imaging [24][25][26]. In the work described in this paper, we have used third harmonic generation imaging which is especially sensitive to fat droplet boundaries to show that the LLS component coincides with the droplets [27].…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Measuring the effect of a Western diet on liver tissue architecture by FLIM autofluorescence and harmonic generation microscopy

Ranjit

Dvornikov

Dobrinskikh

et al. 2017

Biomed. Opt. Express

Self Cite

View full text Add to dashboard Cite

The phasor approach to auto-fluorescence lifetime imaging was used to identify and characterize a long lifetime species (LLS) (~7.8 ns) in livers of mice fed with a Western diet. The size of the areas containing this LLS species depends on the type of diet and the size distribution shows Western diet has much larger LLS sizes. Combination of third harmonic generation images with FLIM identified the LLS species with fat droplets and the droplet size distribution was estimated. Second harmonic generation microscopy combined with phasor FLIM shows that there is an increase in fibrosis with a Western diet. A new decomposition in three components of the phasor plot shows that a Western diet is correlated with a higher fraction of free NADH, signifying more reducing condition and more glycolytic condition. Multiparametric analysis of phasor distribution shows that from the distribution of phasor points, a Western diet fed versus a low fat diet fed samples of mice livers can be separated. The phasor approach for the analysis of FLIM images of autofluorescence in liver specimens can result in discovery of new fluorescent species and then these new fluorescent species can help assess tissue architecture. Finally integrating FLIM and second and third harmonic analysis provides a measure of the advancement of fibrosis as an effect of diet.

show abstract

Section: Data Acquisition: Diver and Phasor Approach To Flimmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Measuring the effect of a Western diet on liver tissue architecture by FLIM autofluorescence and harmonic generation microscopy

Ranjit

Dvornikov

Dobrinskikh

et al. 2017

Biomed. Opt. Express

Self Cite

View full text Add to dashboard Cite

show abstract

“…Tissue phantoms imitating brain optical properties and used in experiments were prepared according to [9,33].…”

Section: Methodsmentioning

confidence: 99%

“…In our microscope light losses are also reduced by means of refractive index matching throughout the optical path. The system has proven effective on imaging of biological and artificial samples, such as murine colon and small intestine, vasculature in the skin, subcutaneous xenograft tumors in mice and various tissue phantoms [6,7,8,9].…”

Section: Introductionmentioning

confidence: 99%

The DIVER Microscope for Imaging in Scattering Media

Dvornikov

Malacrida

Gratton

2019

MPs

Self Cite

View full text Add to dashboard Cite

We describe an advanced DIVER (Deep Imaging Via Emission Recovery) detection system for two-photon fluorescence microscopy that allows imaging in multiple scattering media, including biological tissues, up to a depth of a few mm with micron resolution. This detection system is more sensitive to low level light signals than conventional epi-detection used in two-photon fluorescence microscopes. The DIVER detector efficiently collects scattered emission photons from a wide area of turbid samples at almost any entrance angle in a 2π spherical angle. Using an epi-detection scheme only photons coming from a relatively small area of a sample and at narrow acceptance angle can be detected. The transmission geometry of the DIVER imaging system makes it exceptionally suitable for Second and Third Harmonic Generation (SHG, THG) signal detection. It also has in-depth fluorescence lifetime imaging (FLIM) capability. Using special optical filters with sin-cos spectral response, hyperspectral analysis of images acquired in-depth in scattering media can be performed. The system was successfully employed in imaging of various biological tissues. The DIVER detector can be plugged into a standard microscope stage and used as an external detector with upright commercial two-photon microscopes.

show abstract

“…The ability of two-photon excitation to limit excitation to a defined volume within the plane of focus of the imaging system allows close to diffraction-limited imaging with inherent optical sectioning. Techniques to enhance light collection such as lower numerical aperture (NA) objective lenses to increase the solid angle of collection ( Tung et al., 2004 ; McMullen and Zipfel, 2010 ; Singh et al., 2015 ; Combs et al., 2011 ) and light collection in the transmission plane ( Dvornikov and Gratton, 2016 ) can be employed to take advantage of the fact that the scattered emission arises mainly from the focal plane. Enhancements to the excitation characteristics include imaging within the second infra-red imaging window (>1000–1300 nm) and using regenerative amplifiers to increase laser pulse energy at lower repetition rates.…”

Section: Discussionmentioning

confidence: 99%

Two-photon excitation of FluoVolt allows improved interrogation of transmural electrophysiological function in the intact mouse heart

Salerno

Garten

Smith

et al. 2020

Progress in Biophysics and Molecular Biology

View full text Add to dashboard Cite

Background & aims Two-photon excitation of voltage sensitive dyes (VSDs) can measure rapidly changing electrophysiological signals deep within intact cardiac tissue with improved three-dimensional resolution along with reduced photobleaching and photo-toxicity compared to conventional confocal microscopy. Recently, a category of VSDs has emerged which records membrane potentials by photo-induced electron transfer. FluoVolt is a novel VSD in this category which promises fast response and a 25% fractional change in fluorescence per 100 mV, making it an attractive optical probe for action potential (AP) recordings within intact cardiac tissue. The purpose of this study was to characterize the fluorescent properties of FluoVolt as well as its utility for deep tissue imaging. Methods Discrete tissue layers throughout the left ventricular wall of isolated perfused murine hearts loaded with FluoVolt or di-4-ANEPPS were sequentially excited with two-photon microscopy. Results FluoVolt loaded hearts suffered significantly fewer episodes of atrio-ventricular block compared to di-4-ANEPPS loaded hearts, indicating comparatively low toxicity of FluoVolt in the intact heart. APs recorded with FluoVolt were characterized by a lower signal-to-noise ratio and a higher dynamic range compared to APs recorded with di-4-ANEPPS. Although both depolarization and repolarization parameters were similar in APs recorded with either dye, FluoVolt allowed deeper tissue excitation with improved three-dimensional resolution due to reduced out-of-focus fluorescence generation under two-photon excitation. Conclusion Our results demonstrate several advantages of two-photon excitation of FluoVolt in functional studies in intact heart preparations, including reduced toxicity and improved fluorescent properties.

show abstract

Imaging in turbid media: a transmission detector gives 2-3 order of magnitude enhanced sensitivity compared to epi-detection schemes

Cited by 9 publications

References 26 publications

Measuring the effect of a Western diet on liver tissue architecture by FLIM autofluorescence and harmonic generation microscopy

Measuring the effect of a Western diet on liver tissue architecture by FLIM autofluorescence and harmonic generation microscopy

The DIVER Microscope for Imaging in Scattering Media

Two-photon excitation of FluoVolt allows improved interrogation of transmural electrophysiological function in the intact mouse heart

Contact Info

Product

Resources

About