mitochondrial density analysis

Confocal Microscopic Analysis of Rat Muscle Mitochondria

Mitochondria form highly dynamic networks that are mainly regulated by the balance between its elongation (fusion) and fragmentation (fission)1,2, which are modulated by the expression and activity of proteins such as Mitofusin 1 and 2 (Mfn1 and Mfn2), and Optic protein atrophy 1 (Opa1), which regulate the fusion of the outer mitochondrial membrane and inner membrane, respectively1,2. Dynamin-related protein (Drp1) predominantly regulates mitochondrial fission when it is phosphorylated in the Ser6163.
In skeletal muscle, it has been well established that the mitochondrial network is arranged in structurally well-defined subpopulations based on their proximity to different cell regions (myofibrils, sarcolemma, and nuclei)4,5. Those mitochondria located right beneath the sarcolemma are called subsarcolemmal mitochondria (SSM), those located between the contractile filaments are called intermyofibrillar mitochondria (IMF), and the mitochondrial subpopulation around the nuclei are called perinuclear mitochondria network (PMN). Moreover, it has been suggested that these mitochondrial subpopulations have region-specific functions and are metabolically specialized4,5.
The maintenance of cellular energy homeostasis, which allows metabolic and contractile function, depends to a large extent on the interaction and communication on specific sites through the mitochondrial network (e.g., IMF and SSM interaction)4,6. In addition to mitochondria network interactions, the mitochondrion can also interact with other organelles, forming structural and functional complexes. In this regard, it has been shown that IMF can be located adjacent to the sarcoplasmic reticulum (SR) and in proximity to the Ca2+ release units (CRU), formed by the transverse tubules (TT)7. This fact is relevant due to the role of mitochondrial Ca2+ uptake in regulating ATP synthesis and apoptosis. Recently, a potential role in regulating cytosolic Ca2+ transients has also been suggested8.
TT are invaginations of sarcolemma that have a periodic distribution along the longitudinal axis of cardiomyocytes and skeletal muscle fibers9,10, similar to the IMF distribution5. Changes in the distribution of TT have important physiological implications, given their role in contractile function. However, these changes have been mainly evaluated in cardiomyocytes. Using Fast Fourier Transform (FFT) analysis allows the conversion of periodic signals from the distance domain to the frequency domain, resulting in an FFT spectrum that indicates the frequency and the regularity of the signal11,12,13. Although there is evidence that the organization of the mitochondrial network in skeletal muscle fibers is essential for adaptation to different metabolic conditions, as during regeneration after muscle injury14,15, most analyses are performed qualitatively.
Additionally, given that mitochondrial dysfunction has been associated with several skeletal muscle-related (e.g., disuse atrophy)2 and non-muscle diseases, particularly metabolic diseases, and the associated loss of muscle mass (i.e., atrophy)16, the quantitative evaluation of the mitochondrial network and distribution in skeletal muscle takes relevance. Recently, a significant difference in the longitudinal distribution of mitochondria of gastrocnemius muscle fibers between an obese group (Ob; Zucker fa/fa rats), and a lean group (Lean; Zucker +/+ rats) was identified through FFT17. This study demonstrated the usefulness of the FFT in analyzing the mitochondrial distribution. Therefore, this protocol presents a methodology to study mitochondria in live-skeletal muscle fibers from images obtained by fluorescence confocal microscopy. Mitochondrial density is quantified by background thresholding, and the analysis of longitudinal mitochondrial distribution by FFT analysis is also described. A workflow scheme is presented in Figure 1.

Author Spotlight: Mitochondrial Remodeling in Skeletal Muscle 

Análise da densidade mitocondrial e distribuição longitudinal em fibras musculares esqueléticas vivas de ratos por microscopia confocal

Watch this Scientific Journal Video about Mitochondrial Density Analysis at JoVE.com

Mitochondrial Density Analysis  

Análise de Densidade Mitocondrial

Comece iniciando o software de análise de imagens. Em seguida, abra o arquivo Z-stack e ajuste a orientação para colocar a fibra horizontalmente. Escolha uma região retangular de interesse ou ROI cobrindo a área mitocondrial.Selecione tamanhos que variam de 65 a 90 micrômetros na direção X e tamanhos apropriados na direção Y com base na largura da fibra. Prossiga para duplicar a pilha Z com o ROI selecionado. E salve-o como uma nova pilha Z no formato Tiff.Salve a posição do ROI da pilha original usando a ferramenta ROI Manager. Abra a caixa de diálogo de limite com o atalho, Command+Shift+T. Escolha o algoritmo de limite Otsu e selecione a opção de fundo escuro preto e branco.Observe o histograma da distribuição da intensidade de fluorescência e o valor limite exibido na caixa de diálogo. Aplique o limite à pilha de imagens binárias, escolha Calcular limite para cada imagem e crie uma nova pilha na caixa de diálogo exibida antes de clicar em OK. Salve a pilha gerada com três imagens binárias no formato Tiff. Em seguida, acesse o menu Analisar e selecione Histograma.Na caixa de diálogo Histograma exibida, clique em Sim. E na caixa de diálogo Histograma da pilha, clique em Lista para obter os dados do histograma. Transfira dados de histograma para uma planilha e identifique mito-pixels com um valor de 255.Calcule a densidade mitocondrial dividindo os mito-pixels com o total de pixels e multiplicando por 100. A fibra de rato obeso apresentou menor densidade mitocondrial. Isso foi confirmado pela análise de imagem da pilha.

Research

JoVE Journal

Biology

Mitochondrial Density Analysis

301 visualizações.  Tecnologico de Monterrey. Comece iniciando o software de análise de imagens. Em seguida, abra o arquivo Z-stack e ajuste a orientação para colocar a fibra horizontalmente. Escolha uma região retangular de interesse ou ROI cobrindo a área mitocondrial.Selecione tamanhos que variam de 65 a 90 micrômetros na direção X e tamanhos apropriados na direção Y com base na largura da fibra. Prossiga para duplicar a pilha Z com o ROI selecionado. E salve-o como uma nova pilha Z no formato Tiff.Salve a posi...

Vídeo: Mitochondrial Density Analysis  

Analysis of the Mitochondrial Density and Longitudinal Distribution in Rat Live-Skeletal Muscle Fibers by Confocal Microscopy

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of the mitochondrial network allows healthy mitochondria to meet cellular demands; however, the loss of this capacity has been related to the development or progression of different pathologies. In skeletal muscle, mitochondrial density and distribution changes are observed in physiological and pathological conditions such as exercise, aging, and obesity, among others. Therefore, the study of the mitochondrial network may provide a better understanding of mechanisms related to those conditions.
Here, a protocol for mitochondria imaging of live-skeletal muscle fibers from rats is described. Fibers are manually dissected in a relaxing solution and incubated with a fluorescent live-cell imaging indicator of mitochondria (tetramethylrhodamine ethyl ester, TMRE). The mitochondria signal is recorded by confocal microscopy using the XYZ scan mode to obtain confocal images of the intermyofibrillar mitochondrial (IMF) network. After that, the confocal images are processed by thresholding and binarization. The binarized confocal image accounts for the positive pixels for mitochondria, which are then counted to obtain the mitochondrial density. The mitochondrial network in skeletal muscle is characterized by a high density of IMF population, which has a periodic longitudinal distribution similar to that of T-tubules (TT).&#160;The Fast Fourier Transform (FFT) is a standard analysis technique performed to evaluate the distribution of TT that allows finding the distribution frequency and the level of their organization. In this protocol, the implementation of the FFT algorithm is described for the analysis of the longitudinal mitochondrial distribution in skeletal muscle.

Here, we present a protocol to analyze changes in mitochondrial density and longitudinal distribution by live-skeletal muscle imaging using confocal microscopy for mitochondrial network scanning.

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of ...

Biologia

Rat Gastrocnemius Muscle Fiber Bundle Dissection

Begin by transferring the excised hind limb of a rat to a 60 milliliter Petri dish containing three milliliters of ice-cold Relax solution. Next, carefully lift the achilles tendon with forceps. Using iris scissors, dissect the muscles away from the bone.With a stereo microscope, identify and separate the entire gastrocnemius muscle. Using fine tip forceps, transfer the lateral muscle head to a new Petri dish with ice-cold Relax solution. Gently hold the muscle with forceps on one end and separate the muscle into bundles using micro scissors.Finally, transfer the fiber bundles to a new Petri dish containing two milliliters of ice-cold Relax solution.

Dissecção do feixe de fibras musculares gastrocnêmio de rato

Confocal Live Cell Imaging of Rat Skeletal Muscle Mitochondria

Start by incubating the rat skeletal muscle fibers with TMRE at room temperature for 20 minutes. Next, launch the confocal microscope standard software, and choose the configuration framework. Under the Hardware Configuration dialogue box, select Laser, and check the Helium Neon 543 option.Access the acquisition dialogue box via the Acquire framework, and select the XYZ panel. Then, select the 512x512 format with a 400 hertz speed. Under the displayed pinhole panel, select arbitrary units, and input three area for the pinhole.In the Bean Path settings dialogue box, select a 20 x water immersion objective with a 0.7 numerical aperture, and an emission wavelength window of 576 to 700 nanometers. Choose DD 488543, and set the laser power for the 543 laser at 15%After a 20-minute incubation, change the incubation medium twice. Next, prepare the recording chamber with a 0.15 millimeter boro silicate glass cover slip.Transfer the fiber bundles to the chamber containing 200 microliters of relaxed solution. Utilize the confocal microscope's bright field mode to identify viable fibers for the fluorescent recording of mitochondria. Adjust, gain, and offset by scanning the fluorescence using the Live button.Choose low fluorescent intensity values for a background of nearly zero arbitrary units and a mitochondrial signal between 100 and 200 arbitrary units. Select a pixel size between 150 to 190 nanometers, and adjust the zoom factor in the XY dialogue box to acquire the fiber's full width. In the Z stack dialogue box, set the Z distance starting at a fiber depth of 15 micrometers up to 22 micrometers, and choose a Z step size of 3 micrometers.Click the Start button to acquire the confocal images, and obtain a Z stack composed of three confocal images acquired at 15, 18, and 21 micrometers of depth. The confocal images of an exercised lean wrap muscle fiber show an expected record of skeletal muscle fiber mitochondria with a consistent fiber pattern. In contrast, the obese rat muscle fiber shows substantial alterations in mitochondrial content and distribution.

Imagem Confocal de Células Vivas de Mitocôndrias do Músculo Esquelético de Rato

Start by launching the image analysis software. Next, open the Z-stack file and adjust the orientation to place the fiber horizontally. Choose a rectangular region of interest or ROI covering the mitochondrial area.Select sizes ranging from 65 to 90 micrometers in the X direction and appropriate sizes in the Y direction based on fiber width. Proceed to duplicate the Z-stack with the selected ROI. And save it as a new Z-stack in Tiff format.Save the ROI position from the original stack using the ROI Manager tool. Open the threshold dialogue box with the shortcut, Command+Shift+T. Choose the Otsu threshold algorithm and select the black and white dark background option.Observe the histogram of the fluorescence intensity distribution and the threshold value displayed in the dialogue box. Apply the threshold to the binary image stack, then choose Calculate threshold for each image and create new stack in the displayed dialogue box before clicking OK.Save the stack generated with three binary images in Tiff format. Next, access the Analyze menu and select Histogram.In the displayed Histogram dialogue box, click Yes. And in the Histogram of stack dialogue box, click on List to obtain histogram data. Transfer histogram data to a spreadsheet and identify mito-pixels with a 255 value.Calculate mitochondrial density by dividing the mito-pixels with the total pixels and multiplying with 100. The obese rat fiber presented a lower mitochondrial density. This was confirmed by the stack image analysis.

Mitochondrial Distribution Analysis Using Fast Fourier Transform

Start by launching the open-source platform for biological image analysis. Open the BinDmito-stack and draw a rectangular region of interest with a 256-pixel width and five-micrometer height. Locate a central and a lateral ROI.Obtain the plot profile of the ROIs using the shortcut Command K and transfer the data to a spreadsheet filling columns B and C.Fill column D by selecting the second cell in column D.Navigate to the Home menu, choose Fill, and click Series. Next, select columns and input the calculated delta sampling frequency as the step value and the calculated S as the stop value. Go to the Data menu, click Data Analysis and select Fourier Analysis.Choose the range of data points in column C and the corresponding range in column E.Now, fill column F with the FFT Magnitude using the IMABS function to return the absolute value from the complex number in E, and multiply by 2 over N for normalization. Autofill column F with this formula. Plot the FFT spectrum using the magnitude in F as a function of FFT frequency in D until S.Finally, find the point of the maximum peak and its corresponding FFT frequency.The plot profiles of the selected ROIs show differences in the fluorescents'distribution between the fibers derived from lean and obese rats, as well as ROI variation within the same fiber. In lateral ROIs, the frequency of mitochondrial longitudinal distribution was similar in the fibers derived from lean and obese rats, with higher amplitude in obese rat fiber. In contrast, the central ROI of the obese rat fiber is an example of a critical reduction of the FFT peak when an important alteration of the mitochondrial distribution is present.