Expand this Topic clickable element to expand a topic
Skip to content
Optica Publishing Group
Journal Home About Issues in Progress Current Issue All Issues Feature Issues

Visualization of mitochondria in cardiomyocytes

Virginijus Barzda, Catherine Greenhalgh, Jürg Aus der Au, Steven Elmore, Johannes HGM van Beek, and Jeff Squier

Open Access Open Access

Abstract

The simultaneous detection of third harmonic (THG), and multiphoton excitation fluorescence (MPF) or second harmonic (SHG) from the same focal volume has led us to the development of a nonlinear multimodal microscopic biological imaging tool. The multimodal microscope has been applied for imaging of isolated live cardiomyocytes, and investigation of structural origin of the THG and SHG signals has been performed. By employing the different image contrast mechanisms, differentiation of structures inside a single live adult rat cardiomyocyte has been achieved. Based on structural crosscorrelation image analysis between NAD(P)H fluorescence and THG, and morphology of cardiomyocytes we were able to assign large part of the structure revealed by THG to the mitochondria. The crosscorrelation of THG with fluorescence of tetramethylrhodamine methyl ester (TMRM) labeled cardiomyocytes confirmed the mitochondrial origin of THG. The SHG generated structures were anticorrelated with THG and possessed the characteristic pattern of the myofibrils in the myocyte in accordance with the literature. Possible visualization of mitochondria with THG microscopy appeared due to enhancement of the third harmonic by multilayer arrangement of cristae.

©2005 Optical Society of America

Full Article  |  PDF Article
More Like This
Influence of semicrystalline order on the second-harmonic generation efficiency in the anisotropic bands of myocytes

Catherine Greenhalgh, Nicole Prent, Chantal Green, Richard Cisek, Arkady Major, Bryan Stewart, and Virginijus Barzda
Appl. Opt. 46(10) 1852-1859 (2007)

Photothermal imaging of skeletal muscle mitochondria

Toru Tomimatsu, Jun Miyazaki, Yutaka Kano, and Takayoshi Kobayashi
Biomed. Opt. Express 8(6) 2965-2975 (2017)

Label-free optical imaging of mitochondria in live cells

David Lasne, Gerhard. A. Blab, Francesca De Giorgi, François Ichas, Brahim Lounis, and Laurent Cognet
Opt. Express 15(21) 14184-14193 (2007)

View More...

Supplementary Material (4)

Media 1: AVI (637 KB)     
Media 2: AVI (564 KB)     
Media 3: AVI (775 KB)     
Media 4: AVI (600 KB)     

Cited By

Optica participates in Crossref's Cited-By Linking service. Citing articles from Optica Publishing Group journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (7)
Fig. 1.
Fig. 1. Schematic diagram of the multimodal microscope. F-denotes filters, L-lenses, M-mirrors, DM-dichroic mirrors, and PMT stands for photomultipliers.

Download Full Size | PDF

Fig. 2.
Fig. 2. A sample image of an isolated cardiomyocyte in white light. The rectangular region represents a typical scanning area for the nonlinear multimodal microscope. The length of the entire cardiomyocye shown here is approximately 100 μm.

Download Full Size | PDF

Fig. 3.
Fig. 3. (637KB) Comparison of NAD(P)H MPF and THG images in a typical cardiomyocyte excited with 837nm laser pulses. (a) A 2D optical section of NAD(P)H MPF of a cardiomyocyte; (b) The same 2D optical section simultaneously imaged with THG. The intensity profiles along the same line shown in (a and b) are plotted in (c), where the green diamonds indicate MPF and blue triangles THG intensities. (d) The 2D SCIA output for the slice shown in (a) and (b), red color indicates the correlated region while green and blue are the uncorrelated MPF and THG, respectively. (e) The rendered NAD(P)H MPF. (f) The rendered THG image obtained simultaneously with the MPF. (g) Results of the SCIA where red represents the correlated signal, while green is the uncorrelated NAD(P)H MPF and blue is the uncorrelated THG signal. (h) The same SCIA as (g) but showing only the correlated volume in red. The laser beam propagation is parallel to the z direction while the origin of the axis is placed at the perimeter of the cardiomyocyte. The size of a pixel is 0.24μm

Download Full Size | PDF

Fig. 4.
Fig. 4. (565KB) The comparison of autofluorescence and THG of a typical cardiomyocyte with 1064nm excitation. (a) A 2D slice of the autofluorescence emitted at 630-700nm; (b) The THG generated simultaneously with the MPF. (c) The 3D rendered image of autofluorescence; (d) the rendered image of simultaneously generated THG. (e) The SCIA where red is the correlated signal, while green, which is barely noticeable, is the uncorrelated autofluorescence and blue is the uncorrelated THG signal. (f) The same SCIA as (e) but showing only the correlated volume in red. The laser beam propagation is parallel to the z direction while the origin of the axis is placed at the perimeter of the cardiomyocyte. The size of a pixel is 0.24μm

Download Full Size | PDF

Fig. 5.
Fig. 5. (775KB) Comparison of MPF and THG images of TMRM labeled cardiomyocyte excited with 1064nm laser pulses. (a) A 2D optical section of TMRM MPF of a cardiomyocyte collected at 630-700 nm; (b) The same 2D optical section simultaneously imaged with THG. The intensity profiles along the same line shown in (a and b) are plotted in (c), where the green diamonds indicate MPF and blue triangles show THG intensities. (d) The 2D SCIA output for the slice shown in (a) and (b), red color indicates the correlated region while green and blue are the uncorrelated MPF and THG, respecitively. (e) The rendered 3D image of TMRM MPF. (f) The rendered THG image obtained simultaneously with the MPF. (g) Results of the SCIA where red represents the correlated signal, while green is represents uncorrelated NAD(P)H MPF, and blue is the uncorrelated THG signal. (h) The same SCIA as (g) but showing only the correlated volume in red. The laser beam propagation is parallel to the z direction while the origin of the axis is placed at the perimeter of the cardiomyocyte. The size of a pixel is 0.12μm

Download Full Size | PDF

Fig. 6.
Fig. 6. (600KB) Comparison of SHG and THG images of cardiomyocyte excited with 1064nm laser pulses. (a) A 2D optical section of SHG of a cardiomyocyte; (b) The same 2D optical section simultaneously imaged with THG. (c) The rendered 3D image of SHG. (d) The rendered THG image obtained simultaneously with the SHG. (e) Results of the SCIA where red, which is barely visible, represents the correlated signal, while green represents uncorrelated SHG, and blue is the uncorrelated THG signal. (f) The same SCIA as (e) but showing only the only the minute correlated volume in red. The laser beam propagation is parallel to the z direction while the origin of the axis is placed at the perimeter of the cardiomyocyte. The size of a pixel is 0.24μm

Download Full Size | PDF

Fig. 7.
Fig. 7. Pearson’s coefficient ( r ) analysis of optical slices at different depths of typical cardiomyocytes. Graph shows how THG and MPF are correlated (r >0) through most depths of the sample, while THG and SHG are anticorrelated (r < 0) for much of the sample. In the middle of cardiomyocyte, the coefficient shifts closer to 0, where correlation is difficult to determine.

Download Full Size | PDF

Equations (2)

Equations on this page are rendered with MathJax. Learn more.

r = ∑ A i B i − ∑ A i ∑ B i N ( ∑ A i 2 − ( ∑ A i ) 2 N ) ( ∑ B i 2 − ( ∑ B i ) 2 N )
For a i > a min ∧ b i > b min : C i = a i a max • b i b max ( a i a max • b i b max ) max , otherwise C i = 0
Select as filters
Select Topics Cancel
Top
       
© Copyright 2024 | Optica Publishing Group. All rights reserved, including rights for text and data mining and training of artificial technologies or similar technologies.
Privacy | Terms of Use