ABSTRACT

Light microscopy is a powerful tool that can detect and measure cellular and subcellular structural changes over time. This can be done quickly with transmitted light microscopy without perturbing the cells with stains or probes. For instance, cells undergoing apoptosis often shrink in size and have characteristic blebs in the plasma membrane. Many fluorescent probes/reporters are also used for different phases of apoptosis, such as tagged caspase 3. This paper will showcase the practical applications of several imaging modalities (transmitted light and fluorescence) for detecting apoptosis in endpoint and time-lapse images/sequences.

Address correspondence to: Richard W. Cole, New York State Department of Health, Wadsworth Center, Albany, NY, USA (E-mail: [email protected], Phone: 518-474-7048)

Competing Interests: The authors declare no competing interest.

INTRODUCTION

Programmed cell death is a critical process that regulates cell growth and organism health and can be categorized as either necrosis or apoptosis. Necrosis and apoptosis differ significantly in both mechanism and impact on surrounding cells. Whereas necrosis is caused by external factors such as injury, toxins, or hypoxia (resulting in cell rupture and ensuing inflammation), apoptosis can be initiated by either extrinsic or intrinsic signals such as aberrant mitotic division (aneuploidy), reactive oxygen species (ROS), or triggering of the P450 pathway.[1] The specific mechanisms of apoptosis can vary depending on the cell type, the severity of the stress, and other factors. The process of apoptosis includes cell shrinkage, DNA fragmentation, and the release of signaling nucleotides such as adenosine triphosphate (ATP) and uridine triphosphate (UTP) through the plasma membrane channel pannexin 1 (PANX1), which in turn signals immune cells to engulf the dying cell(s) via phagocytosis—resulting in minimal or no inflammation. Apoptosis is a crucial component of development and tissue renewal and protects the organism by eliminating cells that have undergone aberrant events, which could otherwise lead to genetic mutations and cancer.

Cell death contributes significantly to failed imaging experiments. When conducting live cell imaging, the utmost importance lies in finding the correct conditions to maintain cells in a near-homeostatic condition.[2] This is crucial for observing normal cellular function such as correct chromosomal segregation and cell cycle timing in the presence of synthetic fluorophores and/or fluorescent proteins. Hypoxia, high temperature, and pH changes can all trigger cell death during live cell imaging. Additionally, high photon doses from over-illumination or even illuminating with specific wavelengths, like those in the ultraviolet range, can cause severe cellular damage and changes in cellular processes. Failure to maintain optimal conditions can lead to significant changes in cellular morphology and processes, potentially compromising the accuracy of the research. Therefore, monitoring is essential to ensure that imaging does not induce necrosis or apoptosis.[3],[4],[5]

Methods to monitor cell death include light microscopy, electron microscopy, flow cytometry, electrochemistry, proteomics, and genomics(Table 1) [6],[7],[8],[9],[10],[11],[12]. However, only light microscopy enables real-time observation of cell death. With diverse imaging modalities, light microscopy can easily detect and measure necrosis, apoptosis, aneuploidy, and other structural changes. Transmitted light modalities, such as phase contrast (PC) and differential interference contrast (DIC), allow for visualization of morphological changes such as cytoplasmic blebbing, nuclear fragmentation, and cell shrinking. Fluorescence light microscopy is a modality that enables visualization of fragmented DNA (through the use of DNA binding dyes such as Hoechst, 4′,6-diamidino-2-phenylindole [DAPI], or propidium iodide as well as terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] assay read-out), activation of proteins involved in the apoptosis pathway (i.e., caspase 3/7), and disruptions in membrane integrity (through the use of Annexin V or Biotracker Apo-15).

Intrinsic and extrinsic apoptosis can be experimentally induced by different methods. Staurosporine is a protein kinase inhibitor that induces intrinsic apoptosis through two parallel pathways—caspase dependent and independent. Alternatively, aspirin can induce caspase activation and apoptotic cell morphology by inducing translocation of Bax to the mitochondria and triggering the release of cytochrome c into the cytosol.[13] Here, we use staurosporine to visually demonstrate markers of apoptosis using the most straightforward light microscopy modality, DIC, and the most commonly used, Hoechst or fluorescent-tagged caspase-3/7.

EXAMPLES OF APOPTOSIS DETECTION ASSAYS

• Annexin V is a protein that binds to phosphatidylserines (PS), typically located on the inner leaflet of the plasma membrane and is exposed during the late stages of apoptosis. This protein can be tagged with several types of detection molecules (i.e., fluorescent probes or absorbent stains) and visualized using various methods such as light microscopy or flow cytometry.

• Caspase-3/7 proteases are critical mediators of mitochondrial events of early apoptosis. These proteins can be tagged with different types of detection molecules (i.e., fluorescent probes or absorbent stains) and visualized using various methods such as light microscopy, flow cytometry, or spectrophotometry.

• BioTracker Apo-15 is a fluorogenic peptide that binds to negatively charged phospholipids exposed on apoptotic cell membranes without interfering with cellular function and is visualized by light microscopy or flow cytometry.

• Terminal deoxynucleotidyl transferase (TdT) TUNEL assay uses the enzyme TdT to attach deoxynucleotides to the 3’-hydroxyl terminus of DNA breaks and is visualized by light microscopy.

• DNA ladder assay is used to characterize apoptosis by detecting the ladder formation of fragmented DNA by gel electrophoresis.

One of the advantages of using light microscopy to detect cellular changes is its simplicity. It is possible to detect and monitor cellular changes by viewing the cells in a transmitted light modality, with no staining or significant sample preparation. PC or DIC imaging can differentiate between apoptosis and necrosis. The cytoplasm of necrotic cells swells and does not bleb as compared to apoptotic cells. Three imaging-based techniques (live cell imaging, immunofluorescence [IF]/immunocytochemistry [ICC], and immunohistochemistry [IHC]) provide information regarding apoptosis through visualization of cellular ultrastructure or proteins involved in crucial apoptotic pathways.

Researchers often need to detect the onset of apoptosis to determine causality. This is critical for cancer research because resistance to common apoptotic triggers is frequently described as a hallmark of cancer. Light microscopy is usually the quickest and most efficient detection method and can be done in real time. Here, we describe the advantages of various imaging modalities and provide examples of healthy and apoptotic cells.

METHODS

Cell culture

Stock cultures of the rat kangaroo kidney epithelial cell line PtK (2n = 12), initially purchased from American Type Culture Collection (batch F-10679; Rockville, MD), were grown within 75 cm² T-flasks at 37°C in Eagle’s Minimum Essential Medium (EMEM) + 2 mM L-glutamine + 0.1 mM non-essential amino acids + 1.0 mM sodium pyruvate + 10% fetal bovine serum (FBS). Hela cells, initially purchased from American Type Culture Collection (batch F-19779; Rockville, MD), were grown within 75 cm² T-flasks at 37°C in Dulbecco’s Modified Eagle Medium (DMEM) + 2 mM L-glutamine + 0.1 mM non-essential amino acids + 1.0 mM sodium pyruvate + 10% FBS. For experiments, the cells were enzymatically removed from the T-flasks. They were pipetted into MatTek glass bottom 35 mm Petri dishes (Fisher Scientific, Waltham, MA) containing EMEM media without phenol red indicator for 24 hours before use.

Induction of apoptosis

PtK or Hela cells growing in MatTek dishes were treated with 10 µm Staurospine/1% dimethyl sulfoxide (DMSO [Cat 19-123 Millipore sigma, Bedford, MA]) 30 minutes prior to imaging [5],[14],[15],[16]. Staurosporine is a protein kinase inhibitor that induces apoptosis in mammalian cells through both caspase-dependent and caspase-independent pathways. Staurosporine-induced apoptosis involves mitochondrial caspase activation, which is inhibited by Bcl-2.

Real-time detection of apoptosis

NucView® 488 kit[17] (Biotium, Fremont, CA) was used to monitor apoptosis. The substrate is initially non-fluorescent and capable of penetrating the plasma membrane. During apoptosis, caspase-3/7 cleaves the substrate and releases the high-affinity DNA dye, leading to nuclear fluorescent staining. Visualization of morphological changes in the nucleus during apoptosis is detectable without interfering with the apoptotic pathway.

Time-lapse imaging

Treated cultures (Staurospine or Staurospine + NucView) were imaged in a single Z plane by time-lapse light microscopy with a 2-4 frames/min framing rate using a Nikon Eclipse Ti (Melville, NY) inverted light microscope equipped with DIC, fluorescent optics, and a Perfect Focus system. All microscopy and camera functions were controlled with Element Software. For DIC, cells were illuminated with shuttered, green (GFI filter 510-560 nm) heat-filtered light obtained from 100 W tungsten bulbs. For fluorescence, a white light LED Lumencor Sola (Beaverton, OR) was used as the illumination source with a fluorescein isothiocyanate (FITC) filter cube (480/30). They were viewed and imaged with 60 X (NA 1.40) objective using DIC or fluorescence (FITC). Images were captured with a PCO Edge sCMOS camera (Excelitas Technologies, Waltham, MA). Temperature, CO₂ level (5%), and humidity were controlled with a microscopy incubation chamber (In Vivo Scientific, Salem, SC).

Image processing

ImageJ/Fiji software (https://imagej.net/software/fiji/)[18] was used to adjust the brightness/contrast of the images, and a median filter (radius 3) was applied to allow the plasma membrane to be more easily visualized during the apoptosis process. Selected images from the time-lapse series were used to create montages. Times and scale bars were added for clarity. For image volumes collected in the wide-field modality, blind-iterative deconvolution[19] graphics processing unit (GPU) processing (AutoQuant Media Cybernetics, Rockville, MD) was used to improve the resolution and signal-to-noise of the images. The spherical aberration of the images was measured, and those parameters were used to modify the image stack’s theoretical point spread function (PSF) through the AutoQuant software. This modified PSF was applied in the standard AutoQuant algorithm (30 iterations) to deconvolve the image stack.[20],[21]

3D imaging and display

Fixed cells stained for chromatin visualization (Hoechst 33342 1 µg/ml) were imaged in 3D using a 405 nm LASER (10% power). 3D image stacks (0.1 µM Z spacing) were collected with 63X (NA 1.40) objective on Dragonfly 200 spinning disk confocal (Oxford Instruments, Concord, MA) coupled to a Lecia DM8 inverted microscope (Leica, Deerfield, IL). Images were captured on Zyla sCMOS camera (2Kx2K) (Andor). All microscopy and camera functions were controlled with Fusion Software. The 3D image stacks were visualized using Imaris (Oxford Instruments, Concord, MA).

Fixation and staining

PtK cells were seeded on MatTek dishes (Thermo Fisher Scientific, Waltham, MA) at a concentration of 3.5 x 10⁵ cells. Cells were then treated with an apoptosis-inducing agent, 10 µm Staurospine/1% DMSO, dissolved in DMEM for 4 hours at 37°C. Cells were then washed with PHEM buffer, fixed with 2% glutaraldehyde, and treated with sodium borohydride (NaBH4), and the DNA was stained with 1 µg/ml Hoechst 33342.

RESULTS

Transmitted time-lapse (DIC) imaging can detect both early and late stages of apoptosis. DIC’s low photon dose is critical to ensure that the imaging does not cause apoptosis. Figure 1 shows the progression of 10 µm Staurospine-induced apoptosis over time in Hela cells. The time-lapse montage demonstrates the heterogeneous nature of apoptosis onset; however, all cells eventually undergo apoptosis. The tell-tale signs of apoptosis (e.g., membrane blebbing, nuclear changes, and, finally, cell membrane breakdown) are visible using this method.

Apoptosis can also be tracked by monitoring characterized pathways of cell death, such as the caspase 3 and 7 pathways activated during apoptosis, irrespective of the specific death-initiating stimulus. These proteases are widely considered to coordinate the demolition phase of apoptosis by cleaving a diverse array of protein substrates. There are commercially available real-time probes for monitoring this path, such as the NucView® 488 kit (Biotium, Fremont, CA). Figure 2 shows a montage from a time-lapse series of PtK cells with 10 µm Staurospine-induced apoptosis, imaged in DIC (B&W) and NucView® 488 (green). One of the advantages of monitoring an apoptotic pathway in this manner is earlier detection, which enables the characterization of the early cellular processes contributing to cell death.

Figures 3 and 4 clearly show the contrast between the nuclear structure of control cells and Staurospine-treated cells, which induced the nuclei to undergo apoptosis. While the nuclei in Figure 3 appear normal, the nuclei in Figure 4 show condensation of chromatin, disassembly of nuclear scaffold proteins, and degradation of DNA consistent with the effects of apoptosis.

DISCUSSION

Understanding apoptosis and its detection methods is vital for advancing our knowledge of cell biology and developing effective treatments for various diseases. Apoptosis detection is used to evaluate the efficacy of potential drugs during drug development. Compounds that induce apoptosis in cancer cells or prevent it in healthy cells are particularly interesting. Dysregulation of apoptosis is implicated in a range of diseases, including cancer, neurodegenerative diseases, and autoimmune disorders. By detecting apoptosis, researchers can gain insights into the underlying mechanisms of these diseases and identify potential therapeutic targets.

While there are many methods for detecting and monitoring cell death, light microscopy is the most straightforward, quickest, and least expensive method (given that you can access a light microscope). This approach can also be used to investigate temporal events via time-lapse, light multi-dimensional microscopy, which, combined with apoptotic biomarkers such as NucView, allows for real-time monitoring of cell death.

ACKNOWLEDGMENT

This work was supported by the New York State Department of Health’s Wadsworth Center Advanced Light Microscopy and Image Analysis Core Facility.