Cryo-confocal fluorescence imaging of the milled lamella on the TEM grid after cryo-FIB preparation. Residual fluorescence signal confirms that the targeted region of interest—containing malaria parasite structures—has been successfully retained during lamella preparation, supporting accurate correlation between cryo-fluorescence microscopy and cryo-electron microscopy workflows.

Fluorescence-Guided Cryo-ET of Malaria Parasites

A correlative cryo workflow combining cryo-planing, cryo-fluorescence imaging, and cryo-EM to identify parasite-rich regions before lamella preparation

Cryo-confocal fluorescence imaging of the milled lamella on the TEM grid after cryo-FIB preparation. Residual fluorescence signal confirms that the targeted region of interest—containing malaria parasite structures—has been successfully retained during lamella preparation, supporting accurate correlation between cryo-fluorescence microscopy and cryo-electron microscopy workflows. Milled_lamella_on_the_TEM_grid_after_cryo-FIB_preparation.jpg

Summary

Understanding malaria parasite development in the mosquito midgut is challenging because parasites are distributed heterogeneously within the host tissue. Researchers therefore need workflows that identify biologically relevant regions before high-resolution structural analysis.

This application note presents a correlative cryo workflow designed to address four challenges in generating ultrastructural data from malaria parasites in the mosquito midgut: heterogeneous parasite distribution, limited visibility in cryogenic samples, the risk of missing relevant regions during lamella preparation, and the need to correlate fluorescence and electron microscopy data across multiple preparation steps.

The workflow combines high-pressure freezing of mosquito midguts with EM ICE, cryo-planing with the UC Enuity ultramicrotome, and cryo-CLEM (correlative light and electron microscopy), linking confocal fluorescence images acquired with STELLARIS Cryo to cryo-electron tomography data.

This approach supports targeted preparation and analysis of parasite-containing regions in frozen biological samples and maintains correlation from cryo-fluorescence imaging through high-resolution cryo-electron tomography.

Key learnings

  • Target parasite-rich regions in complex tissue: Fluorescence-guided targeting enables identification of rare parasite-rich regions in frozen mosquito midgut tissue before lamella preparation for cryo-TEM
  • Improve localization with cryo-planing: Cryo-planing improves fluorescence signal clarity for targeting and confocal analysis.
  • Reduce work on non-informative areas: Target localization before cryo-TEM preparation reduces time spent on non-informative areas.
  • Maintain correlation across the cryo workflow: Fluorescence signal is retained after planing and thinning, enabling correlation from STELLARIS Cryo to high-resolution EM.
  • Support analysis of malaria parasite ultrastructure: Cryo-ET reveals parasite morphology, internal structural organization, and host–pathogen interactions.

Introduction

Malaria parasite development in the mosquito midgut is difficult to analyze because parasites are not uniformly distributed within host tissue. 

This correlative workflow combines high-pressure freezing, cryo-planing, cryo-fluorescence imaging, and cryo-EM to identify parasite-containing oocysts before lamella preparation and enable targeted cryo-electron tomography.

Identification of parasite-rich regions before lamella preparation

Mosquito midguts were dissected and transferred into high-pressure freezing planchettes prepared with yeast paste as cryoprotectant. The loaded planchettes were then high-pressure frozen with EM ICE.

DAPI staining before high-pressure freezing was used to visualize nuclei throughout the vitrified tissue and support orientation within the sample.

Video: Cryo-fluorescence overview of a frozen sample carrier containing mosquito midgut tissue stained with DAPI. Regions of increased fluorescence intensity highlight areas with higher cellular density, enabling identification of potential malaria parasite-rich regions prior to cryo-planing and downstream cryo-EM analysis Sample courtesy: Nedal Darif, Postdoctoral Fellow, EMBL Heidelberg, Germany.

Cryo-planing improves the clarity of fluorescent signal in vitrified samples

Under cryogenic conditions, brightfield imaging alone provides limited information for locating internal regions of interest. Cryo-planing and trimming with the UC Enuity ultramicrotome enabled controlled removal of surface material, produced a clean block face, and progressively exposed the target region.

The workflow with the UC Enuity cryo chamber comprised initial block-face visualization by brightfield imaging, iterative cryo-planing with fluorescence inspection, and trimming to expose internal structures. After planing, the fluorescence signal was clearer, supporting more accurate localization of the region of interest.

Comparison of cryo-fluorescence signal before and after cryo-planing of the frozen sample. Controlled removal of surface layers enhances visibility of parasite-associated structures, supporting fluorescence-guided targeting for subsequent cryo-FIB lamella preparation. Sample courtesy: Nedal Darif, Postdoctoral Fellow, EMBL Heidelberg, Germany.
Figure 1: Comparison of cryo-fluorescence signal before and after cryo-planing of the frozen sample. Controlled removal of surface layers enhances visibility of parasite-associated structures, supporting fluorescence-guided targeting for subsequent cryo-FIB lamella preparation. Sample courtesy: Nedal Darif, Postdoctoral Fellow, EMBL Heidelberg, Germany.

Fluorescence imaging guides serial lamella preparation with the SOLIST method

Following planing, cryo-confocal imaging with STELLARIS Cryo was used to visualize parasites within individual oocysts. These oocysts contain developing malaria parasites and define regions of interest for targeted lamella preparation.

Lamellae were prepared from the identified regions on an Aquilos 2 cryo-FIB-SEM using the SOLIST method (Serialized on-grid lift-in sectioning for tomography). The lamellae were transferred onto support grids and thinned to ~200 nm by FIB milling.

Cryo-confocal imaging reveals malaria parasite-containing oocysts in midgut tissue

Cryo-confocal fluorescence imaging of the cryo-planed sample reveals spherical oocysts within mosquito midgut tissue. These structures contain developing malaria parasites and define regions of interest for targeted lamella preparation in the correlative cryo-electron tomography workflow. Sample courtesy: Nedal Darif, Postdoctoral Fellow, EMBL Heidelberg, Germany; Image acquisition: Martin Fritsch, ALMF, EMBL Heidelberg, Germany
Figure 2: Cryo-confocal fluorescence imaging of the cryo-planed sample reveals spherical oocysts within mosquito midgut tissue. These structures contain developing malaria parasites and define regions of interest for targeted lamella preparation in the correlative cryo-electron tomography workflow. Sample courtesy: Nedal Darif, Postdoctoral Fellow, EMBL Heidelberg, Germany; Image acquisition: Martin Fritsch, ALMF, EMBL Heidelberg, Germany.

Targeted cryo-FIB lamella preparation from fluorescence-defined regions

Cryo-FIB-prepared lamellae derived from fluorescence-identified regions of interest. Controlled thinning of vitrified biological samples enables downstream cryo-electron microscopy and tomography for structural analysis of malaria parasites within their native tissue context. Sample courtesy: Nedal Darif, Postdoctoral Fellow, EMBL Heidelberg, Germany; Image acquisition: Zhengyi Yang, Cryo-Electron Tomography Specialist, EMBL Imaging Centre, EMBL Heidelberg, Germany.
Figure 3: Cryo-FIB-prepared lamellae derived from fluorescence-identified regions of interest. Controlled thinning of vitrified biological samples enables downstream cryo-electron microscopy and tomography for structural analysis of malaria parasites within their native tissue context. Sample courtesy: Nedal Darif, Postdoctoral Fellow, EMBL Heidelberg, Germany; Image acquisition: Zhengyi Yang, Cryo-Electron Tomography Specialist, EMBL Imaging Centre, EMBL Heidelberg, Germany.

Cryo-confocal imaging of milled lamella on TEM grid confirms successful targeting

Cryo-confocal fluorescence imaging of the milled lamella on the TEM grid after cryo-FIB preparation. Residual fluorescence signal confirms that the targeted region of interest—containing malaria parasite structures—has been successfully retained during lamella preparation, supporting accurate correlation between cryo-fluorescence microscopy and cryo-electron microscopy workflows. The two images on the left are high-magnification views of two regions within the lamellae shown on the right, used for cryo-electron tomography.
Figure 4: Cryo-confocal fluorescence imaging of the milled lamella on the TEM grid after cryo-FIB preparation. Residual fluorescence signal confirms that the targeted region of interest—containing malaria parasite structures—has been successfully retained during lamella preparation, supporting accurate correlation between cryo-fluorescence microscopy and cryo-electron microscopy workflows. The two images on the left are high-magnification views of two regions within the lamellae shown on the right, used for cryo-electron tomography.

Cryo-ET reveals ultrastructural features of malaria parasites

The prepared lamellae were analyzed by cryo-TEM on a Titan Krios. Cryo-ET revealed parasite morphology, membrane organization, internal structural features, and the surrounding host-tissue context.

Cryo-electron tomography of malaria parasite ultrastructure

Cryo-electron tomography of malaria parasites within a cryo-FIB-prepared lamella, acquired on a Titan Krios transmission electron microscope. The tomographic reconstruction reveals membrane organization and internal structural features, linking fluorescence-guided targeting with high-resolution information from the preserved tissue context.
Figure 5: Cryo-electron tomography of malaria parasites within a cryo-FIB-prepared lamella, acquired on a Titan Krios transmission electron microscope. The tomographic reconstruction reveals membrane organization and internal structural features, linking fluorescence-guided targeting with high-resolution information from the preserved tissue context.

Application value

  • Enables efficient targeting to regions of interest for cryo-ET in complex biological samples
  • Reduces time spent on lamella preparation and cryo-ET in non-relevant regions
  • Supports integration of cryo-fluorescence and cryo-EM workflows
  • Facilitates analysis of rare biological events

Related Articles

Interested to know more?
Talk to our experts.

Price

I need a configuration or price info.

Demo

I need an on site or remote demo.

Service & Repair

I need help keeping my system running: technical service, repairs, spare parts, upgrades or software licenses.

Application Support

I need assistance/training in how to operate my system properly, or how to run a specific application with my system.

Do you prefer personal consulting? Show local contacts

Beckman Coulter LinkBeckman Coulter LogoGenedata LinkIDBS LogoIDBS LinkIDBS LogoAbcam Limited LinkAbcam Limited LogoMolecular Devices LinkMolecular Devices LogoPhenomenex LinkPhenomenex LogoSciex LinkSciex LogoAldevron LinkAldevron LogoIDT LinkIDT Logo
Scroll to top